A Screening for Selected Human Pharmaceuticals in water using spe-hplc, ogun state, Nigeria.

Abstract

Pharmaceuticals are a structurally diverse class of emerging contaminants that have been detected throughout the world as trace contaminants in the water environment. The study is aimed at determining the occurrence and quantification of diclofenac, paracetamol, ibuprofen, ciprofloxacin, sulphadoxine and amodiaquine in well water, tap water and river water. The study is conducted around a hospital environment, in Ogun State, Nigeria, using SPE and HPLC analysis. Water samples were collected from tap water, well water and river water around Olabisi Onabanjo University Teaching Hospital, Sagamu, Ogun State. Samples were extracted using solid phase extraction technique and further analyzed using High Performance Liquid Chromatography. The tap-water water samples contained paracetamol, ibuprofen, diclofenac, ciprofloxacin and sulphadoxine in concentrations of 0.306ng/ml, 3.738ng/ml, 0.138ng/ml, 0.44ng/ml and 1.012ng/ml respectively. The well water samples contained paracetamol, ibuprofen, sulphadoxine and amodiaquine in concentrations of 0.152ng/ml, 5.078ng/ml, 1.008ng/ml and 0.01892ng/ml while the river water samples were found to contain paracetamol, ibuprofen and sulphadoxine in concentration 0.192ng/ml, 3.042ng/ml and 1.294ng/ml respectively. The results confirm pharmaceuticals contamination indeed occurred in the water samples collected, which further supports previous studies around the world. Of significant importance, is the detection of sulphadoxine and amodiaquine waste, which to the best of our knowledge have not been detected elsewhere in the world. Effective water treatment plants that can conveniently remove pharmaceuticals in water is warranted, thus, preserving life and ecosystem at large.

Full text

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AJPSP 2017; Volume 5, Issue 1
Olaitan et al
AJPSP August 15, 2017
A Screening for Selected Human Pharmaceuticals
in Water Using SPE-HPLC, Ogun State, Nigeria
Olaitan O James*1, Chimezie Anyakora2, Ifeoluwa O. Adetifa1, Aderonke A. Adepoju-Bello2
1Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmacy, Olabisi
Onabanjo University, Sagamu, Ogun State
2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Lagos
*Corresponding Author E-mail: olatundeolaitan@hotmail.com
ABSTRACT
Pharmaceuticals are a structurally diverse class of emerging contaminants that have been
detected throughout the world as trace contaminants in the water environment. The study is
aimed at determining the occurrence and quantification of diclofenac, paracetamol, ibuprofen,
ciprofloxacin, sulphadoxine and amodiaquine in well-water, tap-water and river-water. The
study is conducted around a hospital environment, in Ogun State, Nigeria, using SPE and HPLC
analysis. Water samples were collected from tap-water, well-water and river-water around
Olabisi Onabanjo University Teaching Hospital, Sagamu, Ogun State. Samples were extracted
using solid phase extraction technique and further analyzed using High Performance Liquid
Chromatography. The tap-water water samples contained paracetamol, ibuprofen, diclofenac,
ciprofloxacin and sulphadoxine in concentrations of 0.306ng/ml, 3.738ng/ml, 0.138ng/ml,
0.44ng/ml and 1.012ng/ml respectively. The well-water samples contained paracetamol,
ibuprofen, sulphadoxine and amodiaquine in concentrations of 0.152ng/ml, 5.078ng/ml,
1.008ng/ml and 0.01892ng/ml while the river-water samples were found to contain paracetamol,
ibuprofen and sulphadoxine in concentration 0.192ng/ml, 3.042ng/ml and 1.294ng/ml
respectively. The results confirm pharmaceuticals contamination indeed occurred in the water
samples collected, which further supports previous studies around the world. Of significant
importance, is the detection of sulphadoxine and amodiaquine waste, which to the best of our
knowledge have not been detected elsewhere in the world. Effective water treatment plants that
can conveniently remove pharmaceuticals in water is warranted, thus, preserving life and
ecosystem at large.
Keywords:
Pharmaceuticals, Water, Environment, Contaminants

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Introduction:
Until recently, so much time was spent on drug
discovery with little or no time expended at
ascertaining the fate of these drugs after use.
Recently, increasing global concerns over the
public health impacts attributed to
environmental pollution efforts are now being
garnered towards the search of pollutants that
cause health hazards in humans, animals, and
the ecosystem at large. Some of these
pollutants are pharmaceuticals and personal
care products (PPCP). While considerable
effort has been made in developed countries,
African countries seem to be lagging in the
prevention of pharmaceutical waste
environmental contamination (1).
Pharmaceutical products contain active
ingredients that have been designed to have
pharmacological effects and confer significant
benefits to society. They however, become our
enemies when these products find their way
into the environment and cause some
immediate or long-term damages to
microorganisms, plants, animals, man and the
eco-system at large.
While the potential side effects on human and
animal health arising from direct treatment have
been widely documented, only recently has the
implications of the occurrence, fate and effects
of such medicines on the environment have
been considered (2). A range of
pharmaceuticals, including hormones,
antibiotics, NSAIDS, antidepressants and
antifungal agents have been detected in soils,
surface water and ground water (3, 4, 5, 6, 7).
The occurrence of pharmaceuticals in the
environment and the water cycle at trace levels
(in the range of nanograms to low micrograms
per liter) has been widely discussed and still
being published in the literature.
Concentrations of pharmaceuticals in surface
waters, groundwater and partially treated water
are typically less than 0.1 µg/L (or 100 ng/L),
and concentrations in treated water are
generally below 0.05 µg/L (or 50 ng/L) (8). The
increase in detection is largely attributable to
the advances in analytical techniques and
instrumentation. This research is aimed at
detecting the presence of analgesics
(paracetamol, ibuprofen and diclofenac),
antibiotic (ciprofloxacin) and antimalarials
(sulphadozine and amodiaquine) in water.
Paracetamol Ibuprofen Diclofenac
Ciprofloxacin Sulphadoxine Amodiaquine
Figure 1: Chemical Structures of the Studied Compounds

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METHODOLOGY:
CHEMICALS
All chemicals, reagents were of analytical
grade, highest purity and obtained from Fischer
Scientific UK. They included methanol HPLC
grade, acetonitrile HPLC grade, triflouroacetic
acid (TFA) HPLC grade. Standard paracetamol
(BP), ibuprofen (BP), diclofenac (BP),
ciprofloxacin powders (BP), sulphadoxine (BP)
and amodiaquine were supplied by Sigma-
Aldrich (Steinheim, Germany). Solid Phase
Extraction Cartridges i.e. C18, Si-Cyano, C8-
(12ml, 2g) were purchased from SiliCycleInc,
Quecbec Canada.
SAMPLE COLLECTION
Water samples were collected in triplicate from
three different sources (tap-water, river-water
and well-water) at a healthcare institution that
has been in operation for over 25 years in Ogun
State, Nigeria. The sampling was carried out
between December 2013 and January 2014
using coherent protocols and procedures
designed to obtain a representative water
sample. Water samples were collected into
pre-cleaned amber glass-bottles. Samples
were analyzed within 36-hours of collection. To
minimize contamination of samples. Use of
personal care items such as insect repellents,
colognes, and perfumes, caffeinated products,
pharmaceuticals and tobacco were discouraged
during sample collection and processing (9).
SAMPLE PREPARATION
The first step in sample preparation was to
subject water samples collected to a pre-
filtration process by passing the sample through
a 0.45-ųm glass fiber filter. The filtrates were
respectively collected into a clean container.
To further minimize contamination of the
samples. Use of personal care items such as
insect repellents, colognes, perfumes as well as
the use of caffeinated products,
pharmaceuticals and tobacco were avoided
during this process.
SPE EXTRACTION
Solid-phase extraction (SPE) procedures were
employed to extract the target analytes from the
aqueous samples. Water, 5mls, and 5mls of
10% methanol were measured and poured into
each cartridge to activate the sorbents (C18,
C8, Cyano). Water was added to promote the
adsorption of the analytes onto the sorbents.
Water samples, 500mls each, were loaded into
the cartridge at a rate of 10ml/min. The rate at
which each of the water samples was applied
was controlled. Methanol 10mls (10%) was
used as the wash solvent which was poured
into the cartridge to remove sample constituent
that were less retained on the sorbent than the
analyte of interest. Methanol 5mls, (100%)
which was of high eluting strength was poured
into the cartridge, precisely controlled at a rate
of 2ml/min to ensure reproducible result.
PREPARATION OF STOCK SOLUTION OF
STANDARD
A 200 µg/ml concentration stock solution was
prepared for each of the pharmaceuticals using
their respective standards. From the stock
solution, 50µg/ml, 20gµ/ml, 10µg/ml, 5µg/ml
and 1µg/ml concentrations were also made
using serial dilution.
HPLC ANALYSIS
Analyses of the six extracted compounds were
quantitatively carried out using, a Reversed
Phase Agilent 1100 LC System. The analytes
were separated with their respective
chromatographic conditions as stated below
(Table 1:

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Table 1: Chromatographic conditions of the pharmaceuticals.
SP
MP
FR
UvDW
IV
RT
IBUPROFEN
YMC C18
(100 x
4.6 mm,
5
0.1% TFA:
ACN
(40:60)
1.0ml/min
248nm
10
µl
3.8minutes
DICLOFENAC
YMC C18
(100 x
4.6 mm,
5
MeOH
(100%)
0.5ml/min
283nm
10
µl
3.5minutes
PARACETAMOL
YMC C18
(100 x
4.6 mm,
5
NaH2PO4:
ACN
(65:35)
0.8ml/min
260nm
10
µl
2.5minutes
CIPROFLOXACINE
HCL
YMC C18
(100 x
4.6 mm,
5
0.1%
TFA:ACN
(80:20)
1.0ml/min
278nm
10
ml
3.2minutes
SULPHADOXINE
YMC C18
(100 x
4.6 mm,
5
0.1%
TFA:ACN
(70:30)
1.0ml/min
278nm
10
µl
3.8minutes
AMODIAQUINE
YMC C18
(100 x
4.6 mm,
5
0.1%
TFA:MeOH
(10:90)
1.0ml/min
341nm
10
µl
2.0minutes
Key: SP-Stationary Phase, MP-Mobile Phase, FR-Flow Rate, UvDW-UV Detector Wavelength, IV-
Injector Volume, RT-Run Time, TFA-Tetrafluoroacetic Acid, MeOH-Methanol, NaH2PO4- Sodium
Diydrogen-Phosphate, ACN-Acetonitrile

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Results:
Calibration curves were obtained using
standard concentrations of the standards for all
the six pharmaceuticals. The six calibration
curves were all linear with a correlation
coefficient ranging from 0.9882-0.995. All
water samples analysed largely contained the
pharmaceuticals in varying concentrations.
Table 2 below, provides a summary of results
obtained in this study. Further illustrations of
the distribution of the pharmaceuticals in the
water samples are shown in Figures 2 to 10
below.
Table 2: Average concentration of pharmaceutical water samples
Pharmaceutical
Water samples
Water source
Concentration(ng/ml)
Paracetamol
A
B
C
Tap-water
Well
River
0.306
0.152
0.192
Ibuprofen
A
B
C
Tap-water
Well
River
3.738
5.078
3.042
Diclofenac
A
B
C
Tap-water
Well
River
0.138
NOT DETECTED
NOT DETECTED
Ciprofloxacin
A
B
C
Tap-water
Well
River
0.44
NOT DETECTED
NOT DETECTED
Sulphadoxine
A
B
C
Tap-water
Well
River
1.012
1.008
1.294
Amodiaquine
A
B
C
Tap-water
Well
River
NOT DETECTED
0.01892
NOT DETECTED

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Figure 2: Concentration of paracetamol in each of the water samples
Figure 3: Concentration of ibuprofen in each of the water samples
0.306
0.152
0.192
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
Concentration (ng/ml)
A B C
Concentration (ng/ml)
3.738
5.078
3.042
0
1
2
3
4
5
6
Concentration (ng/ml)
A B C
Concentration (ng/ml)

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Figure 4: Concentration of diclofenac in each of the water samples
Figure 5: Concentration of ciprofloxacin in each of the water samples
0.138
0
0
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
Concentration (ng/ml)
A B C
Concentration (ng/ml)
0.44
0
0
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
C oncentration (ng/ml)
A B C
Concentration (ng/ml)

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Figure 6: Concentration of sulphadoxine in each of the water samples
Figure 7: Concentration of amodiaquine in each of the water samples
1.012
1.008
1.294
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Concentration (ng/ml)
A B C
Concentration (ng/ml)
0
0.01892
0
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.018
0.02
Concentration (ng/ml)
A B C
Concentration (ng/ml)

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Figure 8: Concentration of pharmaceuticals found in borehole water sample
Figure 9: Concentration of pharmaceuticals found in well-water sample
0.306
3.738
0.138
0.44
1.012
0
0.5
1
1.5
2
2.5
3
3.5
4
Concentration (ng/ml)
PCM IBU DICLO CIPRO SULPHA
Concentration (ng/ml)
0.152
5.078
1.008
0.01892
0
1
2
3
4
5
6
Concentration (ng/ml)
PCM IBU SULPHA AMD
Concentration (ng/ml)

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Figure 10: Concentration of pharmaceuticals found in river-water sample
Discussion:
The distribution of each pharmaceutical
analyzed in the water samples is shown in
Figures 2 to 7.
Paracetamol was detected in all water
samples (tap-water, well-water, and river-
water) with concentrations of 0.306ng/ml,
0.152ng/ml and 0.192ng/ml respectively as
shown in Figure 2. Figure 3 shows the
distribution of Ibuprofen in each water
sample with concentrations of 3.738ng/ml,
5.078ng/ml and 3.042ng/ml in tap-water,
well and river-water samples respectively.
Diclofenac and ciprofloxacin were detected
only in tap-water water sample with
concentrations of 0.138ng/ml and 0.44ng/ml
respectively as seen in Figures 4 and 5.
Sulphadoxine was detected in all water
samples (tap-water, well-water, river-water)
with concentrations of 1.012ng/ml,
1.008ng/ml and 1.294ng/ml respectively as
shown in Figure 6. Amodiaquine was
detected in well-water only, at a
concentration of 0.01892ng/ml as
demonstrated in Figure 7. In sample A (tap-
water), ibuprofen was observed to have the
highest individual concentration of
3.738ng/ml, with other drug concentrations
observed to be as follows: sulphadoxine
1.012ng/ml, ciprofloxacin 0.44ng/ml,
paracetamol 0.306ng/ml, and diclofenac
0.138ng/ml. Figure 8 demonstrates
distribution of the concentrations of five
pharmaceuticals that were detected in the
borehole sample. In sample B (well-water),
the pharmaceutical with the highest
individual concentration was ibuprofen with
a concentration of 5.078ng/ml, with other
drug concentrations as follows:
sulphadoxine 1.008ng/ml, paracetamol
0.152ng/ml, and amodiaquine
0.01892ng/ml, as demonstrated in Figure 9.
In sample C (river-water), three
pharmaceuticals were found in the sample,
these included: Paracetamol, ibuprofen and
sulphadoxine. Ibuprofen was observed to
have the highest concentration at
3.042ng/ml, with the other drug
concentrations as follows: sulphadoxine
1.294ng/ml, and paracetamol 0.192ng/ml.
Paracetamol, ibuprofen and sulphadoxine
were detected in all the water samples
tested. Ibuprofen was observed to have the
0.192
3.042
1.294
0
0.5
1
1.5
2
2.5
3
3.5
Concentration (ng/ml)
PCM IBU SULPHA
Concentration (ng/ml)

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highest concentration in all the water
samples that were analyzed with
concentrations of 3.738ng/ml, 5.078ng/ml
and 3.042ng/ml in tap-water water, well-
water and river-water respectively. The
average concentration of paracetamol
detected in all water samples analyzed was
0.2167ng/ml, average concentration for
ibuprofen in all water samples was
3.9526ng/ml, while the average
concentration for sulphadoxine was
1.1046ng/ml.
The presence of paracetamol, diclofenac,
ibuprofen and ciprofloxacin in water
samples from Sango Ota, Ogun State,
Nigeria is in line with an earlier study in this
environment which showed concentrations
of µg/ml (6). In a similar previous research
by Kolpin et al. (6), most concentrations
recorded exceeded 1 µg/ml. Chronic
exposure to diclofenac can impair renal
functions in fish. The kidney has also been
found to be a target organ of diclofenac
toxicity in many organisms such as birds,
mice and humans (6,9,10,11). The
exposure of activated sludge microbes to 5-
500 μg/L of diclofenac, ibuprofen, can lead
to a shift in the community structure and
inhibit the growth of bacteria (genus
Nitrospira) that play a key role in nitrification
during wastewater treatment (12). These,
may in turn, reduce the removal efficiency of
NSAIDs in wastewaters. Laboratory-based
experiments showed that ibuprofen was
fully mineralized by microbes present in
activated sludge but only after addition of
lactose as another carbon source, a
mechanism known as co-metabolism (13).
The effective diclofenac concentration for
chronic fish toxicity was in range of
wastewater concentrations. Diclofenac
residues and renal disease were
reproduced experimentally in oriental white-
backed vultures by direct oral exposure and
through feeding vultures the remains of
diclofenac-treated livestock (9).
The anti-inflammatory compounds,
ibuprofen (up to 93 ng/l) and diclofenac (up
to 261 ng/l) were among the most frequently
detected. In a survey conducted by the
United States Geological Survey. Ibuprofen
was detected at a maximum concentration
of 1.0 ug/l (0.20 ug/l median concentration,
0.018 ug/l reporting level) at a 9.5%
frequency in 84 submitted water samples
from a network of 139 US stream sampling
sites across 30 states during the period of
1999 to 2000 (4). Ciprofloxacin, for
example, was detected in concentrations
between 0.7 and 124.5 µg/L in hospital
effluent.
Information about the effects of the active
substances on organisms in aquatic and
terrestrial environments is increasing but
still too little. Effects on fish, daphnia,
algae, and bacteria have been
demonstrated using low concentrations in
long-term tests. Ciprofloxacin, for example,
was found in concentrations between 0.7
and 124.5 µg/L in hospital effluent (14).
Bacteria resistance to antibiotics has been
observed in the aquatic environment (15).
The links between the presence of
antimicrobials and the favoring of resistant
bacteria as well as the transfer of resistance
at concentrations as low as those found for
antimicrobials in the environment have not
yet been established. This could also
explain the resistance in antimalarial-
therapy facing this part of Africa. The result
from this research further supports
observations and conclusions of a number
of previous studies outside Nigeria that
suggests pharmaceutical compounds are
present in water which include the presence
of ibuprofen in Somes river, Romania, in
concentrations of 300-10000ng/l (16);
ibuprofen and diclofenac detected in Pearl
river, South China, in concentrations of 17-
685ng/l (17); ibuprofen and paracetamol
detected in Nairobi river, Kenya, in
concentrations of 10 -30µg/l (18).

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Conclusion:
This research demonstrates water samples
(tap-water, well-water and river-water)
obtained from Olabisi Onabanjo University
Teaching Hospital Sagamu, Ogun state
contain six pharmaceutically active
ingredients. These include, paracetamol,
ibuprofen, diclofenac, ciprofloxacin,
sulphadoxine, amodiaquine in varying low
concentrations. Due to low concentrations
of these pharmaceuticals, their health
impact on humans may be minimal. The
accumulation of these pharmaceutical
agents over time can pose some harmful
effects such as antimicrobial resistance,
toxicity in humans, as well as aquatic
toxicity. Further investigations of
pharmaceuticals in African waters is
therefore warranted.

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