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Journal of Planar Chromatography 27 (2014) 4, 287–293 DOI: 10.1556/JPC.27.2014.4.9 287
0933-4173/$ 20.00 © Akadémiai Kiadó, Budapest
Summary
Two methods were described for the simultaneous determination of
ciprofloxacin HCl (CIP) and moxifloxacin HCl (MOX) in their bina-
ry mixture present in industrial wastewater. A solid-phase extrac-
tion procedure (SPE) based on retention on HLB OASIS cartridges
and elution with a mixture of methanol–water in acidic medium was
preformed, and then both fluoroquinolones were separated using
two chromatographic methods. The first method was based on high-
performance liquid chromatographic separation of the two drugs
on reversed-phase Zorbax C18 column. The mobile phase consisted
of monobasic potassium phosphate (50 mM, pH 2.5, adjusted with
phosphoric acid) and acetonitrile (80:20, v/v). Flow rate was 1 mL
min
−1
. Quantitation was achieved with ultraviolet (UV) detection at
278 nm. Linearity was found to be over the concentration range of
1–50 µg mL
−1
for both CIP and MOX. The second method was based
on the thin-layer chromatographic (TLC) separation of the two
drugs followed by densitometric measurements of their bands at
278 nm. The separation was carried out on silica gel 60 F
254
plates,
using methanol, ammonia, and methylene chloride (55:35:20, v/v) as
a developing system. The linearity was found to be in the range of
0.25–2.5 µg band
−1
for both CIP and MOX. Both methods were opti-
mized and validated as per International Conference on Har -
monization (ICH) guidelines. Separation was developed on spiked
water samples and checked on process wastewaters of industrial ori-
gin after SPE sample pretreatment.
1 Introduction
Pharmaceutical compounds are widespread contaminants of the
aquatic environment. Since traditionally they have not been
viewed as environmental contaminants, the study of their pres-
ence in the environment is in some ways a new area of research
which has taken in recent years. Our current knowledge indi-
cates that residues of pharmaceuticals at trace quantities are
widely spread in aquatic systems [1]. Antibiotics constitute a
large group of pharmaceuticals, which are widely administered
in human and veterinary medicine. The extensive use of these
antibiotics may result in their presence in the environment.
Antibiotics are believed to be of greatest concern among all
pharmaceuticals due to the potential risk of antibiotic resistance.
Studies in the United States of America and Europe have detect-
ed antibiotic resistant bacteria in drinking water supplies [2, 3].
According to previous studies and publications, one of the most
prevalent groups of antibiotics found in the environment, and
particularly in surface waters, is that of the widely used, highly
potent fluoroquinolones (FQs) [4–6]. Administered FQs are
largely excreted as unchanged compounds in urine, and conse-
quently discharged into hospital sewage or municipal waste-
water. Despite lots of studies with positive detection of antibi-
otics and other pharmaceuticals in soils and environmental
waters and despite of their negative effects on human health,
there is no defined limit value for the occurrence of these pollu-
tants in soils or natural waters.
Therefore, more monitoring and surveillance studies are needed
at local level to determine exactly how the antibiotics make their
way into public waterways and to obtain a better understanding
of the transport and environmental fate of antibiotics.
Ciprofloxacin hydrochloride (CIP) [1-cyclopropyl-6-fluoro-1,4-
dihydro-4-oxo-7-(1-piperazinyl)-3-quinolinecarboxylic acid]
and moxifloxacin hydrochloride (MOX) [1-cyclopropyl-7-
[(1S,6S)-2,8-diazabicyclo[4.3.0]non-8-yl]-6-fluoro-8-methoxy-
4-oxo-quinolone-3-carboxylic acid] are fluoroquinolone anti -
bacterial agents which are active against a wide range of bacte-
ria. Their chemical structures are shown in Figure 1.
Different methods are available for the determination of the
selected FQs, CIP and MOX in environmental water samples.
Water samples are analyzed after solid-phase extraction, by
high-performance liquid chromatography (HPLC) with ultravio-
let (UV) detection [7], fluorescence detection (FD) [8, 9], mass
[10] spectrometric [11], tandem mass spectrometric detection
[12], high-performance liquid chromatography (UPLC) with
diode-array detection (DAD) fluorescence and mass spectrome-
F.I. Khattab and S.M. Riad, Analytical Chemistry Department, Faculty of
Pharmacy, Cairo University, Kasr-El Aini Street, 11562 Cairo, Egypt; and
H. Salem and H.T. Elbalkiny, Analytical Chemistry Department, Faculty of
Pharmacy, October University for Modern Sciences and Arts (MSA), 11787
6th October City, Egypt.
E-mail: heba_elbalkiny@hotmail.com
Determination of Fluoroquinolone Antibiotics in Industrial
Wastewater by High-Pressure Liquid Chromatography and
Thin-Layer Chromatography–Densitometric Methods
Fatma I. Khattab, Hesham Salem, Safaa M. Riad, and Heba T. Elbalkiny*
Key Words
Ciprofloxacin hydrochloride
Moxifloxacin hydrochloride
High-performance liquid chromatography
Thin-layer chromatography–densitometry
Industrial wastewater
try detection [13–15], UPLC tandem mass spectrometry
[16–18], and capillary electrophoresis [19].
The aim of this work was to develop simple, accurate, and sen-
sitive HPLC and TLC–densitometric methods for the simultane-
ous determination of the selected FQs, CIP, and MOX in indus-
trial wastewater.
2 Experimental
2.1 Apparatus
2.1.1 For HPLC Method
HPLC Agilent 1200 series, vacuum degasser, thermostated col-
umn compartment G1316A/G1316B, multiple wavelength
detector, quaternary pump (Germany), and chromatographic
column of Zorbax ODS (4.6 cm × 250 mm, 5 μm) were used.
2.1.2 For TLC–Densitometric Method
CAMAG (Muttenz, Switzerland) TLC instrumental set-up con-
sisting of sample applicator Linomat V, 100-µL syringe
(Hamilton, Switzerland) and TLC Scanner III operated by
winCATS software (V 3.15, CAMAG, Switzerland) were used.
Precoated silica gel 60 F
254
plate (20 × 10 cm
2
) (Fluka Chemie,
Buchs, Switzerland) with 200 µm thickness was employed.
A UV lamp – short wavelength 278 nm was employed for detec-
tion of bands.
2.1.3 Solid-Phase Extraction
SPE apparatus consisted of a 12-port vacuum manifold with
drying attachment and 12 large volume samplers.
2.2 Material
2.2.1 Samples
Ciprofloxacin HCl and moxifloxacin HCl powders were kindly
supplied by the Egyptian Pharmaceutical Industrial Company
(EPICO, Egypt) and El-Rowad Company (RIPC, Egypt),
respectively, and their percentage purities were found to be
100.1 ± 0.95 and 99.99 ± 0.58, respectively, according to official
British Pharmacopoeia (BP) methods [20].
2.2.2 Chemicals
Acetonitrile, methanol, and water of HPLC grade; methylene
chloride and ammonia of analytical grade; potassium dihydro-
gen orthophosphate; and orthophosphoric and ultra-pure sulfu-
ric acids were obtained from Merck (Darmstadt, Germany).
Sodium chloride and ethylenediaminetetraacetic acid (EDTA)
disodium salt were obtained from El Nasr Company (Cairo,
Egypt).
Membrane filters 0.45 µm from Teknokroma (Barcelona, Spain)
were used. The cartridges used for SPE were Oasis HLB (6cc
200 mg) (Waters Corp., Milford, MA, USA).
2.3 Standard Solutions
2.3.1 Stock Solutions
CIP and MOX were prepared by dissolving 100 mg of each
powder in 100 mL of distilled water, forming a solution with a
final concentration (1 mg mL
−1
).
2.3.2 Working Solutions
Working solutions of both CIP and MOX were freshly prepared
by dilution from their stock solutions with mobile phase to
obtain a concentration of 100 μg mL
−1
for CIP and MOX for
HPLC method and diluting with distilled water to obtain con-
centration of 250 μg mL
−1
for TLC–densitometric method.
2.4 Procedure
2.4.1 Collection and Preparation of Samples
A total of six wastewater samples were collected from pharma-
ceutical industries at different sites as shown in Table 1 and
placed in 2-L amber glass bottles. Water samples were cen-
trifuged at 4500 rpm for 5 min to remove possible solid materi-
al and stored in the dark at 4°C [14, 21]. Fluoroquinolones were
concentrated from water samples by solid-phase extraction
method.
2.4.2 Extraction and Clean-Up (SPE Procedure)
After adjustment of the pH to 4.0 with sulfuric acid (1 M) and
addition of 186 mg of EDTA dipotassium salt, 1 L of the sample
was percolated through the Oasis HLB 6 cc (200 mg) cartridge.
The cartridge was previously conditioned with 5 mL of
methanol and 4 mL of ultrapure water. The washing step was
performed with ultrapure water at pH 4.0. Then the cartridge
was eluted with 4 mL of methanol. This eluate was evaporated
to dryness under a gentle stream of nitrogen and the residue was
redissolved in 0.5 mL of mobile phase, and filtered through
0.45 μm Millipore membrane filter (Billerica, MA). The injec-
Determination of FQ Antibiotics in Industrial Wastewater by HPLC and TLC–Densitometry
288 Journal of Planar Chromatography 27 (2014) 4
Table 1
Location of wastewater collection areas.
Samples Location
Industrial wastewater 1, 2 Pharmaceutical company located in
Obour city
Industrial wastewater 3, 4 Pharmaceutical company located in
10th of Ramadan city
Industrial wastewater 5 Pharmaceutical company located in
El Sawah
Industrial wastewater 6 Pharmaceutical company located in
October city
Figure 1
The chemical structures of (a) CIP and (b) MOX.
tion volume of eluate was 20 µL in case of HPLC while dis-
solved in methanol in case of TLC–densitometry.
Because of the zwitterionic nature of FQs, SPE of their analytes
is expected to be strongly pH-dependent. HLB can be used in
both acidic and in basic solutions. For these considerations,
samples were usually acidified to pH 3 to reduce biological
activity, cartridges conditioning and sample loading were done
at pH 3 [22–25].
2.4.3 HPLC Method
2.4.3.1 Chromatographic Conditions
Chromatographic separation of the binary mixture was per-
formed using an isocratic elution of a mobile phase consisting of
sodium dihydrogen phosphate (50 mM, pH 2.5, adjusted with
phosphoric acid) and acetonitrile (80:20, v/v). The mobile phase
was filtered through 0.45-µm membrane filter and degassed for
30 min in an ultrasonic bath prior to its use. The mobile phase
was pumped through C18 column at a flow rate of 1 mL min
−1
.
Analyses were performed at ambient temperature, and detection
was carried out at 278 nm. The injection volume was 20 µL.
2.4.3.2 System Suitability
Twenty microliters of the working solutions were injected. The
system suitability parameters, retention time, tailing factor, the-
oretical plate count (N), height of theoretical plate (HETP), sep-
aration of eluted peaks (resolution), and column capacity were
calculated and compared to the reference United States
Pharmacopeia (USP) guidelines [26].
2.4.3.3 Construction of Calibration Curves
Aliquot volumes (0.1–5 mL) of CIP and MOX working
solutions (100 μg mL
−1
) were transferred separately into 10-mL
measuring flasks, diluted to the volume with the mobile phase.
Twenty microliters of these solutions were injected in triplicate
into the HPLC system. The chromatographic conditions were
applied, and the chromatograms were recorded. Calibration
curves relating to the peak areas ×10
−3
of CIP and MOX versus
their concentration were plotted, and the corresponding regres-
sion equation was calculated.
2.4.4 TLC–Densitometric Method
2.4.4.1 Chromatographic Conditions
TLC aluminum sheets, 20 × 10 cm, precoated with 0.25 mm sil-
ica gel 60 F
254
were used. The samples were applied as bands
(band width: 6 mm, bands were spaced 1 cm apart from each
other and 1.5 cm from the bottom edge of the plate). Linear
ascending development was done in a chromatographic tank
previously saturated with methylene chloride–methanol–ammo-
nia (55:35:20, v/v) for 1 h at room temperature to a distance of
approximately 8 cm from the lower edge. The developed plates
were air dried and scanned at 278 nm on CAMAG TLC Scanner
3 operated in the absorbance mode, with deuterium lamp as a
source of radiation; the slit dimension was kept at 3 mm ×
0.45 mm, and 20 mm s
−1
scanning speed was employed.
2.4.4.2 System Suitability
Parameters including resolution (R
s
) and peak symmetry were
calculated and compared to the reference USP guidelines
[26].
2.4.4.3 Construction of Calibration Curves
Aliquots (1–10 mL) of CIP and MOX working solution (250 µg
mL
−1
) equivalent to 250–2500 µg were accurately transferred
into a series of 10-mL volumetric flasks, and the volumes were
made up to the mark with distilled water to give final concentra-
tions of 25–250 µg mL
−1
. Aliquots (10 µL) of each concentration
equivalent to 0.2 –2.5 µg were applied to the TLC plates as
bands. Calibration curves relating to the peak areas ×10
−3
of CIP
and MOX versus their concentration were plotted, and the cor-
responding regression equations were calculated.
2.4.5 Assay of Laboratory-Prepared Mixtures
Different aliquots of the drugs were accurately transferred from
their working solutions and mixed to prepare solutions of differ-
ent ratios. The chromatographic conditions of both methods
were adopted for each laboratory-prepared mixture, and the con-
centrations of each drug were calculated from the corresponding
regression equation. Each concentration was conducted from the
average of three experiments.
2.4.5 Spiked Water Samples
Samples of pure water spiked with different concentration of
CIP and MOX were treated with previous SPE procedure, then
the adopted chromatographic conditions were applied, and the
recovery of each drug was calculated.
3 Results and Discussion
The aim of this work was to develop two simple chromato-
graphic methods for the simultaneous determination of CIP and
MOX in industrial wastewater. In both methods, we overcome
the problem of the interference of impurities by choosing the
proper clean-up procedure and mobile phase.
3.1 HPLC Method
Improved resolution of both components was achieved using a
mobile phase composed of sodium dihydrogen phosphate buffer
adjusted at pH 2.5 with orthophosphoric acid and acetonitrile
(80:20, v/v).
To optimize the HPLC method, it was necessary to test the
effect of different variables. In order to separate the two drugs
from each other. Two types of stationary phases were tried
(Zorbax C8 and Zorbax SB-C18 columns), but the latter
showed a more suitable resolution. Several ratios of buffer
solution and acetonitrile were checked. Increasing the ratio of
acetonitrile slightly caused some broadening for both peaks.
Using methanol instead of acetonitrile was not successful for
the separation of CIP and MOX. The pH of the mobile phase is
a major factor influencing the chromatographic behavior of
FQs; thus, different PHs were examined. Best peak shape and
adequate separation of the two drugs were obtained by a final
composition of sodium dihydrogen phosphate buffer adjusted
at pH 2.5 with orthophosphoric acid and acetonitrile 80:20
(v/v) as shown in Figure 2.
The calibration curves for both drugs were constructed between
peak area × 10
−3
at 278 nm versus the corresponding concentra-
Determination of FQ Antibiotics in Industrial Wastewater by HPLC and TLC–Densitometry
Journal of Planar Chromatography 27 (2014) 4 289
tion for both drugs, and linear relationships were obtained in the
range of 1–50 µg mL
−1
and for both CIP and MOX.
The parameters of system suitability of this method were com-
pared to reference values [27]. The results are listed in Table 2.
Results obtained by applying the HPLC procedure showed that
the method is valid for the simultaneous determination of CIP
and MOX in the presence of each other in the laboratory-pre-
pared mixtures with mean percentage recovery of 100.63 ± 0.57
and 100.9 ± 0.5 for CIP and MOX, respectively (Table 3).
3.2 TLC–Densitometric Method
The TLC–densitometric method offers a simple way for direct
separation on TLC plate, followed by measuring the optical den-
sity of the separated bands of CIP and MOX at 278 nm.
Studying the optimum parameters for maximum separation was
carried out by trying different developing systems with different
ratios, but complete separation of both drugs was achieved by
using methanol, ammonia, and methylene chloride (55:35:20, v/v)
as a mobile phase, which gave good resolution, sharp, and sym-
metrical peaks. Different scanning wavelengths were tried; on
using 278 nm, the separated peaks were more sharp and symmet-
rical with minimum noise. The R
F
values were as follows: CIP
(0.43 ± 0.02) and MOX (0.54 ± 0.02) as shown in Figure 3a.
The parameters of system suitability of the TLC–densitometric
method were compared to reference values [26]. The results are
listed in Table 2.
The calibration curves were constructed by plotting the integrat-
ed peak area ×10
−3
at 278 nm versus the corresponding concen-
tration of both drugs. Linear relationships were obtained in the
range of 0.25–2.5 μg band
−1
for both CIP and MOX.
Results obtained by applying the TLC–densitometric procedure
showed that the method was valid for the simultaneous determina-
tion of CIP and MOX in the presence of each other in the laborato-
ry-prepared mixtures, with mean percentage recoveries of 100.5 ±
0.64 and100.46 ± 0.73 for CIP and MOX, respectively (Table 3).
3.3 SPE
When an analyte is present at low concentration in complex
environmental samples, such as wastewaters, extraction and
preconcentration must precede chromatographic analysis. In
this work, solid-phase extraction with HLB extraction cartridges
was used for sample preparation. The extraction recoveries of
the analysts were estimated using spiked water samples.
Antibiotics were extracted from water adjusted at pH 4.0. They
were eluted from the disks with methanol. The spiked samples
were extracted in triplicate and analyzed by HPLC–UV and
TLC–densitometry as shown in Figures 2b and 3b, respectively.
The recovered amount was calculated as peak area. Extraction
recoveries for all investigated antibiotics are given in Table 4.
4 Method Validation
Method validation was performed with all the proposed methods
as follows:
4.1 Range and Linearity
The linearity of both methods was evaluated by processing a
minimum of 6-point calibration curves on three different days.
Determination of FQ Antibiotics in Industrial Wastewater by HPLC and TLC–Densitometry
290 Journal of Planar Chromatography 27 (2014) 4
Figure 2
HPLC chromatogram of (a) laboratory-prepared mixture showing the
separation of CIP at (t
R
= 2.296 min) and MOX at (t
R
= 5.311 min) using
the applied chromatographic conditions, (b) the determination of CIP
and MOX in spiked wastewater after solid-phase extraction using the
applied chromatographic conditions, and (c) the determination of
MOX in wastewater sample.
Table 2
Statistical analysis of parameters required for system suitability of HPLC and TLC–spectrodensitometric methods.
Parameter RP-HPLC method TLC–densitometric method Reference value [25, 27]
CIP MOX CIP MOX
t
R
(RP-HPLC) 2.2 5.32 (0.43 ± 0.02) (0.54 ± 0.02) t
R
> 1 (HPLC)
R
F
(TLC)
N(column 7096 13,960 N> 2000
efficiency) Increases with efficiency
of the separation
HETP 0.002 0.001 The smaller the value,
(height equivalent the higher the column
to theoretical plates) efficiency
T(tailing factor) 1.03 0.89 1 1 T< 2
T= 1 for symmetric peak
R
s
(experimental 7.3 1.66 R
s
> 1.5
resolution)
The corresponding concentration ranges, calibration equations,
and other statistical parameters for both methods are listed in
Table 5.
4.2 Limits of Detection and Quantification
The limit of detection (LOD) and limit of quantification (LOQ)
were calculated, respectively, for both drugs using the proposed
methods with a ratio of 3.3 and 10 standard deviations of the
blank and the slope of the calibration line (Table 5).
4.3 Accuracy
To study the accuracy of the proposed methods, procedures
under study of linearity, for both drugs using the proposed meth-
ods, were repeated three times for the determination of five dif-
ferent concentrations of pure CIP and MOX. The accuracy
expressed as percentage recoveries is shown in Table 5. Good
accuracy of the developed methods was indicated by the results
obtained.
4.4 Precision
The precision of the proposed methods, expressed as RSD, was
determined by the analysis of three different concentrations of
pure CIP and MOX within the linearity range. The intra-day pre-
cision was assessed from the results of three replicate analyses
of three pure samples CIP and MOX on a single day. The inter-
day precision was determined from the same samples analyzed
on three consecutive days. The results of intra-day and inter-day
precisions are illustrated in Table 5.
4.5 Specificity
Specificity was ascertained by analyzing different mixtures con-
taining both drugs in different ratios as listed in Table 3. The
separated drugs in the prepared mixtures were confirmed by
comparing their retention times and/or R
F
values to those of stan-
dard solutions and the increase in peak response after spiking of
standards in tested wastewater. Other parameters such as resolu-
tion, capacity factor, and selectivity for the separated spots and
peaks were calculated.
Determination of FQ Antibiotics in Industrial Wastewater by HPLC and TLC–Densitometry
Journal of Planar Chromatography 27 (2014) 4 291
Table 3
Determination of CIP and MOX in laboratory-prepared mixtures by applying the proposed methods.
Mix- Ratio RP-HPLC method TLC–densitometric method
ture CIP MOX CIP MOX
No. Taken Found Recovery % Taken Found Recovery % Taken Found Recovery % Taken Found Recovery %
(μg mL
−1
) (μg mL
−1
) (μg mL
−1
) (μg mL
−1
) (μg mL
−1
) (μg mL
−1
) (μg mL
−1
) (μg mL
−1
)
1 1:1 2 2.01 100.54 2 1.99 99.54 0.8 0.803 100.32 0.8 0.798 99.77
2 1:2 2 1.99 99.69 4 4.03 100.69 0.4 0.404 100.98 0.8 0.797 99.65
3 1:3 1 1.01 100.80 3 2.99 99.66 0.2 0.203 101.43 1.6 1.610 100.63
4 1:5 1 1.01 101.18 5 5.00 100.01 0.2 0.201 100.32 1 1.009 100.93
5 2:1 4 4.04 100.93 2 2.01 100.59 1.2 1.197 99.76 0.6 0.608 101.32
Mean ± SD 100.63 ± 0.57 100.9 ± 0.5 100.5 ± 0.64 100.46 ± 0.73
Figure 3
TLC chromatogram of (a) (1) laboratory-prepared mixture of 0. 5
μg band
–1
of CIP and (2) 0.25 μg band
–1
MOX using methylene chlo-
ride–methanol–ammonia (55:35:20, v/v) as developing system, (b)
spiked wastewater after solid-phase extraction of (1) 0.75 μg band
–1
of
CIP and (2) 2.75 μg band
–1
MOX, and (c) wastewater sample showing
the detection of MOX at R
F
value of 0.48.
4.6 Robustness
For HPLC method, the robustness was investigated by the
analysis of samples under a variety of experimental conditions,
such as small changes in the pH (3.0–3.5) of the buffer in the
mobile phase. The effect on retention time and peak parameters
was studied. It was found that the method was robust when the
mobile phase ratio was varied. During these investigations, the
retention times were modified; however, the areas and peaks
symmetry were conserved.
For TLC–densitometric method, the robustness was investigated
by the analysis of samples under a variety of experimental condi-
tions, such as small changes in proportions of different compo-
Determination of FQ Antibiotics in Industrial Wastewater by HPLC and TLC–Densitometry
292 Journal of Planar Chromatography 27 (2014) 4
Table 4
Mean percent recoveries of FQs in spiked water using the proposed chromatographic methods.
Fluoroquinolones For HPLC method For TLC–densitometric method
Spiked level (mg mL
−1
) Recovery % Spiked level (mg mL
−1
) Recovery %
CIP 2.5 (LOQ) 87.1 0.35 (LOQ) 81.6
50 92.2 2.5 89.3
MOX 2.5 (LOQ) 89.5 0.3 (LOQ) 79.6
50 91.1 2.5 91.5
Table 5
Assay parameters and validation sheet for the proposed chromatographic methods.
Parameters TLC–densitometric method HPLC method
CIP MOX CIP MOX
Wavelength (in nm) 278
Calibration range
a)
0.25–2.5 1–50
LOD
a)
0.110 0.101 0.849 0.843
LOQ
a)
0.335(8.375 µg L
−1
) 0.305 (7.625 µg L
−1
) 2.572 2.555
Slope 0.1314 0.092 0.0973 0. 0.016
Intercept 0.2588 0.1786 0.0013 0.0866
Mean %
b)
100.45 99.89 98.87 100.34
% RSD 1.737 1.413 1.209 1.658
Accuracy
c)
99.02/0.72 100.60/0.75 100.73/0.97 101.2/0.80
Intra-day precision
c)
100.08/0.70 100.25/0.65 100.57/0.61 100.2/0.39
Inter-day precision
c)
100.04/0.64 100.12/0.23 100.21/0.46 100.2/0.46
Robustness
c)
100.64/0.63 99.63/1.45 99.47/0.93 99.75/1.41
Correlation coefficient (r) 0.9995 0.9996 0.9999 0.9999
a)
RP-HPLC methods: in (μg mL
−1
); TLC–spectrodensitometric methods: in (μg band
−1
)
b)
Average of three experiments
c)
Mean value/relative standard deviations (RSD) of three samples
Table 6
Occurrence of pharmaceuticals in industrial wastewater samples by HPLC and TLC–densitometric methods (mg L
–1
).
HPLC method TLC–densitometric method
CIP MOX CIP MOX
W.W1 N.D. N.D. N.D. N.D.
W.W2 N.D 11.25 N.D. 11.3 (0.452 µg band
−1
)
W.W3 1.82 N.D. 1.8 (0.0728 µg band
−1
) N.D.
W.W4 4.13 N.D. 3.92 (0.156 µg band
−1
) N.D.
W.W5 N.D. N.D. N.D. N.D.
W.W6 N.D. N.D. N.D. N.D.
N.D. = not detected
nents by up to ±0.5% mainly of the organic parts of the mobile
phase. The effect on R
F
values and peak parameters was studied. It
was found that the method was robust when the mobile phase
ratio was varied. During these investigations, the R
F
values and
peak symmetry were of minor change; however, the areas were
conserved. The effects of robustness are shown in Table 5.
5 Application of Method
The developed and validated analytical method was applied to
the determination of the target drugs in wastewater samples
from different pharmaceutical industries; a total of six samples
of industrial wastewater were analyzed under the previous con-
ditions described.
Thousand milliliters of prefiltered and acidified (with sulfuric
acid (1 M), pH 4) wastewater samples were applied to Oasis
HLB cartridges. Analytes were eluted with 4 mL of methanol.
This eluate was evaporated to dryness under a gentle stream of
nitrogen, and the residue was redissolved in 0.5 mL of mobile
phase in case of HPLC and methanol in case of TLC–densito-
metric method. Chromatograms of industrial wastewater sam-
ples are shown in Figures 2c and 3c.
The procedures also were applied for industrial wastewater after
spiking with known concentration of both standards to check the
presence of either drug in concentration less than their LOQs.
Identification of target compounds was done by comparison of
TR, R
F
values of pharmaceutical standards, and compounds in
wastewater samples; the results are stated in Table 6.
6 Conclusion
CIP and MOX were extracted and preconcentrated from spiked
water samples by solid-phase extraction (SPE) and quantitative-
ly determined by the two proposed methods. This procedure
resulted in good recoveries for the antibiotics used in this study.
Both proposed methods provided simple, accurate, and repro-
ducible quantitative analysis of both CIP and MOX in laborato-
ry-prepared mixtures and wastewater samples. The HPLC
method was more rapid and gave better resolution between both
drugs, thus lowering analysis time and providing high sensitivi-
ty and selectivity.
Also the TLC–densitometric method had the advantage over the
reported method of using a simpler developing system and was
applied for the quantitative estimation of the cited drugs [28].
The method was found to fulfil the validation requirements of
the analytical methodology for the determination of CIP and
MOX in industrial wastewater.
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Ms received: December 12, 2013
Accepted: April 11, 2014
Determination of FQ Antibiotics in Industrial Wastewater by HPLC and TLC–Densitometry
Journal of Planar Chromatography 27 (2014) 4 293
