CSAWAC 44 (8) 909-1084 (2016) · Vol. 44 · No.

8 · August 2016

CLEAN
Soil Air Water
Renewables
Sustainability
Environmental Monitoring

8 | 2016 www.clean-journal.com
 967

Muhammet Altunok1,2
Armin Ko € nig2
Research Article
Waleed M.M. Mahmoud Ahmed3,4
Tarek Haddad4,5 Automated Determination of Sulfadiazine in Water,
 G. Vasconcelos4,6
TibiriSc a
Klaus Ku € mmerer4
Fish Plasma and Muscle by HPLC with On-Line
Column-Switching
1
Department of Aquaculture, Faculty
_
of Fisheries, University of Izmir Katip Fast, cheap and simple HPLC methods have been gaining importance in the field of
S
Celebi, Turkey
2 antibiotic residue analysis especially in the context of environmentally friendly and
Department of Environmental Health
Sciences, University Medical Center sustainable aquaculture. An effective column-switching technique for HPLC using
Freiburg, Freiburg, Germany online extraction column was developed to eliminate the time consuming pre-
3
Department of Pharmaceutical treatment procedures and to facilitate the high throughput analysis of sulfadiazine in
Analytical Chemistry, Faculty of fish plasma and muscle as well as in water samples. The standard curves for the
Pharmacy, Suez Canal University,
Ismailia, Egypt
determination of sulfadiazine residues from biological matrices and water have good
4
Faculty of Sustainability, Sustainable
linearity (r2 > 0.996). The average recovery ranged from 92.2 to 118% with the relative
Chemistry and Material Resources, standard deviations of 0.3–9.3%. Limits of detection were determined as 5.7 mg L
1 for
Institute of Sustainable and water, 8.1 mg L
1 for plasma and 9.6 mg kg
1 for muscle, while limits of quantification
Environmental Chemistry, Leuphana were 18.9 mg L
1 for water, 26.9 mg L
1 for plasma and 31.9 mg kg
1 for muscle,
University of Lu€neburg, Lu€ neburg,
Germany respectively. Intra- and inter-day precisions were 0.3–9.3 and 0.4–6.7%, respectively.
5
Faculty of Pharmacy, Department of With the retention time of 12 min and total analysis time less than half an hour, this
Pharmacology, University of Aleppo, method provides an effective online extraction process and good sensitivity in liquid
Aleppo, Syrian Arab Republic chromatography, and can be applied for the determination of residual sulfadiazine in
water, fish plasma and muscle at lower than maximum residue level of 100 mg kg
1 fish.
6
Departamento de Quimica da
Universidade Federal de Santa
Maria, Campus Camobi, Santa Maria Keywords: Antibiotics; Aquaculture production; On-line clean up; Plasma; Polystyrene-
RS, Brazil divinylbenzene copolymer
Received: December 1, 2014; revised: September 10, 2015; accepted: March 29, 2016
DOI: 10.1002/clen.201400863

1 Introduction half-life and potential residues, induction of resistant strains in

Sulphonamides represent the most important group of antibiotics
that are widely used in human and veterinary medicine including
aquaculture. Sulfadiazine (SDZ), a sulphanilamide derivative, is a
broad-spectrum antibiotic used in veterinary prophylaxis of infections
and in treatment of diseases as well as growth promoter and feed
additive [1]. Furthermore, it has long been used in the aquaculture
industry worldwide [2–4]. The main route for the antibiotic use in
aquaculture is medicated feed (surface-coated with antibiotic) because
it minimises handling and stress. However, it is not possible to ensure
that individual fish receives the right dose of medication and the risk is
that antibiotics are washed off the feed before it is eaten. The analysis
of SDZ in fish blood is of clinical importance in addition to residual and
pharmacokinetic studies [5, 6]. Long term or heavy use may lead to the
presence of unwanted residues in edible tissues of aquaculture fish [7].
Previous studies reported that sulphonamides showed relatively long
Correspondence: Dr. M. Altunok, Faculty of Fisheries, Department of
_
Aquaculture, University of Izmir S
Katip Celebi, Turkey
E-mail: muhammet.altunok@ikc.edu.tr
Abbreviations: ACN, acetonitrile; DVB, divinylbenzene; LOD, limit of
detection; LOQ, limit of quantification; MRL, maximum residue limit;
PS, polystyrene; RA-MIP, restricted access-molecularly imprinted
polymer; RSD, relative standard deviation; SDZ, sulfadiazine; SPE,
solid phase extraction.
pathogenic bacteria and potential carcinogenic effect, allergies and
toxic effects for human health [8, 9]. There is also concern regarding
residues of this drug in food stuff because of their potential
carcinogenic character. Sulphonamides in the environment are also
toxicological and regulatory concerns. Since they inhibit the growth of
microorganisms, these chemotherapeutic agents become hazardous if
they occur in aquatic environment [10]. Thus, they are on the priority
list of aquaculture drugs and chemicals scheduled for developing
analytical methods in USA and Europe [11]. To minimise the risks to
human health from sulphonamide residues in food, many countries
have established a maximum residue limit (MRL) of 100 mg kg
1 in
edible animal tissues [9].
Several methods [12–14] have been used for the determination of
SDZ in a variety of matrices, most of which are based on liquid
chromatography [15–21]. Most of these methods used in biological
matrices such as blood and tissues require solid phase extraction
(SPE) and several sample preparation processes, like filtration,
deproteinisation, extraction, centrifugation and concentration [22].
Simple and on-line clean-up method as a cost and labour-saving
alternative plays an important role in routine analysis of numerous
environmental samples of which most are time-consuming and
 _
Present address: Izmir S
Katip Celebi €
Universitesi, € unleri Fak€
Su Ur€ ultesi,
35260, Ci _
S gli, Izmir, Turkey.

© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.clean-journal.com Clean – Soil, Air, Water 2016, 44 (8), 967–974
968 M. Altunok et al.
require large volumes of samples, solvents, etc. [23, 24]. There are
several previous reports issued on rapid screening of drugs in serum
and muscle after deproteinisation and extraction of the samples
[22, 25–28]. In a recent review, the applicability of column-switching
HPLC systems for trace analysis of organic compounds was described
by Fernandez-Ramos et al. [29]. The columns packed with restricted
access media, RAM-BSA or restricted access-molecularly imprinted
polymers (RA-MIP) have been used to simplify HPLC analysis of
sulphonamides [30, 31] but there are still time-consuming, labour-
intensive and costly reagents in packing of these columns. In a
recent research, sophisticated preparation of RA-MIP column and
application in column-switching HPLC system was reported for the
analysis of sulphonamides in milk [32]. However, it may not be
practical in many laboratories particularly in developing countries
because of limited laboratory facilities and knowledge on prepara-
tion of restricted access materials and techniques. To the best of our
knowledge, to date, such an on-line extraction by column-switching
HPLC has not been studied extensively in fish plasma and muscle
with small injection volumes (20 mL).
The purpose of this study was to establish a simple, cheap and fast
reliable online clean-up HPLC method using a pre-column packed
with polystyrene-divinylbenzene copolymer (PS/DVB) for determina-
tion of SDZ in fish plasma and muscle. The method was applied to
water samples because of concerns related to veterinary drug
residues in aquatic environment.
2 Materials and methods
2.1 Reagents
Acetonitrile (ACN) (HPLC grade), formic acid (analytical grade) and
SDZ were obtained from Merck (Darmstadt, Germany). Water was
purified with a Milli-Q system from Millipore (Molsheim, France).
2.2 Fish dosing and sample preparation
Rainbow trouts (Oncorhynchus mykiss) obtained from a commercial
fish farm were kept in ponds. Sulfadiazine was administered to two
groups of ten fish in each group orally through the medicated feed
route at two different doses of 50 and 100 mg kg
1 fish daily for ten
days. All medicated fish were collected at the end of ten days (3–6 h
post dosing) and the fish were killed by a blow to the head. Blood
(min. 0.5 mL) was collected immediately from the caudal vein of each
fish. The obtained plasma (isolated by centrifugation) and the whole
filleted fish were kept frozen (
20°C) until analysis. Plasma and
muscle from incurred fish were analysed and the amounts of SDZ in
these samples were calculated by extrapolation from the standard
curves of their respective spiked blank samples. Drug-free (blank)
plasma and muscle tissues were sampled from the control fish,
which are received directly from the fish farm, and they were
processed and kept as described above until use.
2.3 Medicated fish samples
The plasma from medicated fish was thawed and vortexed to assure
homogeneity prior to injection. Incurred fish muscle (1 g) was mixed
in organic solvent (10 mL ACN) for 5 min with an Ultra-Turrax
homogenizer. After centrifugation (4000 rpm at 4°C for 10 min) the
organic layer was pipetted into autosampler vials and injected
directly to HPLC. Water samples obtained from several rivers were
© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.clean-journal.com
only centrifuged before using. The autosampler was set at an
injection volume of 20 mL.
2.4 Calibration standards
The SDZ stock solution (10 mg L
1) was prepared by dissolving in
ACN/water (15:85, v/v) because solubility of SDZ is poor in water and
it was stored protected from light at þ4°C. Calibration standards for
different matrices were prepared by spiking the blanks with the
stock solution at the spiking levels of 0.1–3 mg L
1 for water and
plasma, 0.1–3 mg kg
1 for muscle. Plasma standards were prepared
after thawing the blanks. Spiked plasma was vortex-mixed and after
centrifugation, the supernatant was used directly. Blank muscle
piece was spiked and homogenised in a centrifuge tube, and was
allowed to stand at room temperature in dark for approximately
30 min prior to further processing so that the SDZ was in sufficient
contact with the matrix. After centrifugation, the supernatant was
used in direct injection without any pre-treatment or solid phase
extraction. Calibration standards for water were prepared by
spiking, vortex mixing and centrifugation of the water samples,
then the standards were directly injected. All spiked samples were
finally kept at
20°C in the dark until analysis. Quantification
(linearity, validation) was carried out by using matrix-matched
standard calibrations.
2.5 Equipment
The HPLC-system (Shimadzu, Duisburg, Germany) consisted of three
pumps (LC-10AD, LC-10AT and LC-10AS), a valve (FCV-11AL,
Shimadzu) for selecting mobile phase, a communications bus
module (CBM-20A, Shimadzu), a Uniflow Degasys DG 1310 degasser
(VDS optilab, Berlin, Germany), a UV-vis detector set at 267 nm (SPD-
10AVP, Shimadzu) and an autosampler (SIL-10ADVP, Shimadzu). Two
six-port switching valves (FCV-12AH, Shimadzu) were used for the
automated column switching. The column oven (CTO-10AC,
Shimadzu) was set at a constant temperature of 35°C. Data
acquisition was done on a Shimadzu LC-solution Software v1.2.
2.6 Columns
A Nucleosil C18 column (250  4.0 mm id, 5 mm particle size and
100 Å pore size) preceded by a guard column 8/4 100–5 C18ec, (both
from Macherey-Nagel, Du € ren, Germany) was used as an analytical
column to separate the analyte from matrix interferences. Various
columns were tested (C4, C8, C18, ADS, CN, NH2, LiChrospher1 and
CN, NH2, OH, SA, NUCLEOSIL1) as an extraction column but there
was no success to retain SDZ in these extraction columns to achieve
good recovery. Therefore, the extraction material (Chromabond1
Easy) was obtained from Macherey-Nagel (Du € ren, Germany) and was
dry packed in a stainless steel column cartridge (30  4.6 mm id).
This material is a polar, bifunctionally modified PS/DVB copolymer
with a weak anion exchanger, (specific surface 650–700 m2 g
1,
particle size 80 mm, pore size 50 Å , pH stability 1–14). Before packing,
the empty cartridge was cleaned in an ultrasonic methanol bath.
2.7 Column-switching liquid chromatography
A column-switching HPLC system was used for the determination of
sulfadiazine in water, plasma and tissue. A stainless steel column
(30  4.6 mm) packed with PS/DVB was used as a pre-column for sample
Clean – Soil, Air, Water 2016, 44 (8), 967–974

Figure 1. Column-switching system.
pre-treatment and coupled with the analytical column. The column-
switching system and positions are illustrated in Fig. 1. The valves work
in two different positions: Position 1 (injection/clean-up position) and
position 2 (elution/analysis position). To optimise the online extrac-
tion efficiency, several solvents were tested as a mobile phase. These
includes: different concentration of formic acid in water (0.01–0.5%, v/
v) as well as mixtures of formic acid in water (0.1%, v/v) and acetonitrile
with proportions of 0–70% (Fig. 1, position 1). Furthermore, different
flow rates (1–4 mL min
1) and different duration times (1–10 min)
were applied in order to achieve optimal conditions for the complete
© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.clean-journal.com
Water 969
elution of the plasma and muscle matrix in a possible shortest time
while the target analyte was still adsorbed and eluted selectively in a
subsequent elution step. Three different mobile phases were used; (A)
0.1% formic acid in water for both chromatographic separation and
equilibration; (B) ACN (100%) for both chromatographic separation
and cleaning of extraction column and (C) formic acid (0.1%) in water/
ACN (12:88, v/v) for extraction.
In position 1, the system starts for equilibration of the pre-column
and the analytical column (C18-Nucleosil) with mobile phase A
(2 mL min
1) and mobile phases A and B (A/B, 90:10, v/v, 1 mL min
1),
Clean – Soil, Air, Water 2016, 44 (8), 967–974
970 M. Altunok et al.

respectively. After the injection of the sample, the pre-column was
washed with mobile phase C for 5 min at a flow rate of 1 mL min
1
(pre-treatment step, Fig. 1). By switching to position 2 (Fig. 1), SDZ
was eluted from the pre-column by back-flash and separated on the
analytical column using mobile phases A and B which were run in
gradient until reaching the final ratio of 30:70 v/v at a flow rate of
1 mL min
1. Then the system returned to position 1 automatically
for cleaning and re-conditioning and new sample injection. The
linearity was obtained under the same conditions and procedures
for the calibration standards. The method was validated for
linearity, accuracy, recovery and precision based on spiked stand-
ards and medicated fish samples.
3 Results
3.1 Analytical performance
3.1.1 Linearity
The responses of the studied SDZ HPLC area were linear with six
calibrators over a concentration range of 0.1–3 mg L
1 for each
sample. Matrix matched calibrations were used for validation and
quantification purposes to avoid variations in the analysis of real
samples. Linearity was tested for each matrix by separate calibration
standards in five replications per data point and the coefficient
results are summarised in Table 1. The calibration curve was linear
with the correlation coefficients of R2 > 99.6% in all working
matrices. Typical chromatograms of SDZ in plasma, muscle and
water are shown in Figs. 2 and 3.
3.1.2 Accuracy and precision
Accuracy and precision of the method were studied by spiking blank
samples at four different concentrations (10, 20, 50, 100 mg L
1) using
three replicates per concentration. The values obtained during the five
consecutive days intra- and inter-day precision and accuracy are
summarised in Table 1. Accuracy was evaluated through recovery
studies and the results which were obtained from all matrices with the
average recoveries between 92 and 118% were quite satisfactory. The
recovery experiments showed the highly efficient online clean-up for
fish plasma and muscle. The method exhibited also good precision
experiments resulting in low intra-day relative standard deviations
(RSDs) for all matrices (RSD < 9.3%). Inter-day precision values which
were studied on spiked blank samples at the same concentration
levels, also indicated low inter-day RSDs (<6.7%). RSD values for the
intra- and inter-day precision confirmed the good repeatability and
reproducibility of the method.
3.1.3 Limits of determination and quantitation
The limit of detection (LOD) and limit of quantification (LOQ) were
calculated using the equations:
LOD ¼ 3s=S ð1Þ
and:
LOQ ¼ 10s=S ð2Þ
where s is the standard deviation of intercept, S is the slope [33]. The LOD
for plasma and water was <8.1 mg L
1, while the LOQ did not exceed
26.9 mg L
1 (Tab. 1). The evaluated LOD and LOQ values were 9.6 and
31.9 mg kg
1 in fish muscle, respectively. All values of the quantitation
where within recommended limits under MRL (100 mg kg
1) and were
acceptable for muscle samples (relative standard deviation <10% with
n ¼ 5)
3.2 Analysis of treated fish samples
The method was then applied to real samples from rainbow trout
medicated with SDZ. Control samples from untreated fish were
analysed and no residue was detected. Figure 2 shows the
chromatogram of the muscle sample from medicated fish (100 mg
kg
1 daily). Mean residue levels obtained from three replicates are
indicated in Table 2. Matrix matched calibration was applied for
quantification and determination of SDZ levels in the samples of
medicated fish. The levels varied between 5.2 and 9.6 mg kg
1 in
muscle on the last treatment day and were higher than the MRL
established by the European Union. The sensitivity of this method is
supposed to be sufficient, since the studied dosages are the typical
dose of SDZ (50–100 mg kg
1 per day) in treatment of fish [34] and
other animals [16]. It is also practical for routine and high volume
analysis as the method does not include complex sample pre-
treatments and the determination can be easily performed.
4 Discussion
HPLC analysis of matrix with high protein content usually requires a
multistep clean-up procedure. To shorten and simplify sample pre-
treatment, various methods and columns or extraction cartridges
have been developed and used for direct injection of biological
fluids. Column-switching technique in previous studies showed
excellent efficiency for online clean-up of human plasma samples in
determination of antifungal drugs [35–37]. The column (C18 ADS,
LiChrospher1) was functioned with those analytes as an online

Table 1. Validation parameter of the proposed method for various matrices (calculations for water and plasma in mg mL
1, for muscle in mg kg
1)

Recoverya) (%) Precisionb) (%)

Matrix R2 10 20 50 100 10 20 50 100 LOD LOQ

a) c)
Water 0.999 118 92 98 101 0.3 (4.0) 1.1 (2.0) 2.8 (1.9) 1.8 (0.4) 5.7 18.9
Plasma 0.999 96 101 101 100 7.1 (9.8) 1.9 (2.7) 0.7 (0.9) 0.8 (0.9) 8.1 26.9
Muscle 0.996 108 107 92 101 3.9 (9.8) 1.9 (6.2) 9.3 (6.7) 1.4 (2.5) 9.6 31.9
a)
n ¼ 5.
b)
Precision expressed as relative standard deviation (%) and inter-day precision is given in brackets.
c)
n ¼ 25.

© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.clean-journal.com Clean – Soil, Air, Water 2016, 44 (8), 967–974

extraction column in previous studies for washing human plasma
using water (formic acid 0.1%, v/v) as mobile phase at a flow-rate of
1 mL min
1 [36, 37]. However, SDZ, representing the high-polarity of
the sulphonamide antibiotics, showed no retention on that
extraction column. Also, other columns tested during method
development failed to retain this slightly hydrophilic compound [38]
in the online-extraction (Fig. 1 position 1). Thus, a new SPE material
(PS/DVB, Chromabond1 Easy) which is suitable for a broad range of
© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.clean-journal.com
Water 971
Figure 2. Typical chromatograms of (a) blank (plasma),
(b) spiked water (10 mg mL
1), (c) medicated fish muscle
(100 mg kg
1), and (d) medicated fish plasma
(50 mg kg
1).
compounds (acidic, neutral, basic, polar and nonpolar substances)
because of its polar based retention mechanisms, was used in
packing of the pre-column and tested in the online sample
extraction for the determination of SDZ in water and fish. Due to
the bifunctional modification, the extraction material is consider-
ably more hydrophilic than conventional PS/DVB polymers and thus
easily wettable with water. This polymeric material with a weak ion
exchanger provides a wide range of pH stability (1–14), protection of
Figure 3. Typical chromatograms of spiked (a) water
(20 mg L
1), (b) plasma (30 mg L
1), and (c) fish muscle
(40 mg L
1).
Clean – Soil, Air, Water 2016, 44 (8), 967–974
972 M. Altunok et al.
Table 2. Determination of SDZ in medicated fish plasma and muscle
Precision (%)
Matrix Dosage Measured content Intra Inter
(mg kg
1) (mg L
1 or day day
mg kg
1)
Plasma 50 28.9 1.0 4.7
100 65.3 0.6 2.2
Muscle 50 5.2 0.8 3.9
100 9.6 2.3 8.9
Precision expressed as relative standard deviation (%), n ¼ 5.
the phase from acidic or basic solvents, and its high surface means
very high binding capacity (nearly five times higher than silica-based
adsorbents). Therefore, the material has a phase which is highly
effective for small molecule retention and for hydrophilic layer
which is non-adsorptive for proteins. The method was optimised
further by manipulating solvents with variation of pH and ratio of
water/acetonitrile as well as flow rates of mobile phases. Then the
material provided automated clean-up without any extensive pre-
treatment for direct injection of 20 mL sample volume which was
enough for retention of SDZ on the material and well washing of
interfering components simultaneously.
The on-line solid-phase extraction with HPLC using MIP was
described previously in large volume sample injection (100 mL) for
pre-concentration and analysis of sulphonamides in milk [32]. The
study demonstrated that small sample volumes from environmen-
tal, food and biological matrices can be retained on-line in the
column-switching system which requires only small modifications
to hardware and instruments. A comparison of sample size,
processing time, sensitivity to contamination and cost between the
direct injection and the conventional SPE based techniques was
also reported previously [39]. As previously reported by these
authors, smaller sample sizes will also allow more practical and
cheaper analysis in biological matrices. The results supported that
only a few microliters of water sample can be supplied for analysis
in the case of online SPE. This improvement would make it possible
for rapid assessment of environmental impact and food safety in
aquaculture operations. Most of analytical methods for aquacul-
ture products include different extraction and clean-up procedures
have been developed in order to minimise matrix effects [40]. These
procedures comprise the potential for residual contamination
whereas the online extraction system reduces the contact with
surfaces because there is no need to reuse so much equipment,
which may contain residues from other samples. In the online SPE,
risk for cross-contamination could exist as the column was re-used
but contamination was not observed in the present study.
LOQ, the intra- and inter-day precision and the mean recovery
were in the same range for the developed analysis method of water,
fish plasma and muscle. Also, the results of calibration curves
showed high recovery and validation performances for all matrices
used in the study but it is also important to note that the method
requires the using matrix matched standards. The validation data
obtained reflects the efficiency of the online extraction and also a
less complex analytical procedure. In addition, the validation
period of the proposed method was run on the same pre-column
without repacking or regeneration, demonstrating the reusability
© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.clean-journal.com
of the packed pre-column after an automated reconditioning prior
to each analysis. Therefore, it should also be noted that the method
may be more practical for numerous and high sample throughput
in the laboratory. Also, the reusability of the extraction column
makes the total analysis procedure more rapid and cheaper as the
extraction column had good life time and at least 250 biological
samples were analysed without evidence of changes in its
performance (e.g. high pressure). The pre-packed column was
cleaned with methanol in an ultrasonic bath, when the working
pressure highly increased, and then it was reused in experiments.
During the study, the analytical column was used for more than
500 unprocessed plasma and muscle samples with the same
performance and quite constant peaks showed that probable
damage to the column was not very serious.
The described column switching method using extraction column
was applied first time that allows fast and automated determination
of SDZ in fish plasma and fish tissue samples without any pre-
separation procedures for HPLC. The developed method has two
main steps: firstly, sample clean-up by a pre-column packed with
extraction material (Chromabond1 Easy) that enables separation of
the analyte from the samples. Automation of this step allowed direct
injection and repetition of extraction. Secondly, the chro-
matographic separation is performed through an analytical column
followed by identification with a UV detector. The proposed method
reduces the workload, costs, required amount of sample as well as
time and chemical use per analysis. Additionally, the method, which
provides rapid and simple extraction procedure, represents opera-
tive alternative with realising the on-line monitoring for routine
works in laboratories where basic HPLC equipment is available. The
switching method was described with two valves because of
establishing configuration which is providing high flexibility for
the analysis of various drugs in the laboratory. As an alternative,
when pump C and the autosampler were connected to port 6 of
valve-2 and the pumps A and B to port 3 of valve-2, valve-1 would not
be necessary.
5 Concluding remarks
Pharmaceutical pollution in aquaculture and different environ-
mental matrices is a great public concern because of health risks and
sustainability, and demonstrate the need for improved and fast
analytical methods. The results described in this paper showed that
online extraction was successfully applied to determine SDZ in
plasma and residue levels in edible tissues. Additionally, the method
was also tested with water samples and showed practically good
results which promise to make possible the rapid screening of
antibiotic residues in both water and wild living organisms from
aquaculture surrounding environments. The rapid analysis time
(<20 min including sample preparation) may help to design high-
throughput aquacultural and environmental monitoring. Further-
more, it provides relatively cheap process, requires smaller sample
sizes, and has less contamination risks with equipment. Still, a
number of improvements can be studied in order to lower the
method detection limits, which can be achieved using further
optimisation of mass spectrometric conditions and preparation of
mass-labelled surrogate spiking standards. Consequently, improve-
ment of the method for lowering the LODs, LOQs and for analysis of
aquatic plants, sediments and organism other than fish can be next
research target as they are also under threat from the potential
hazards of aquacultural antibiotics.
Clean – Soil, Air, Water 2016, 44 (8), 967–974

Acknowledgements
The authors would like to thank for research fellowships (grant no:
1939B050900016) from the “Deutsche Forschungsgemeinschaft”
(DFG) and “The Turkish Council of Higher Education” that allowed
to work on the development of the method.
The authors have declared no conflict of interest.
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