Arabian Journal of Chemistry (2017) 10, S719–S729

King Saud University

Arabian Journal of Chemistry

www.ksu.edu.sa
www.sciencedirect.com

ORIGINAL ARTICLE

Analysis of some pharmaceuticals in municipal

wastewater of Almadinah Almunawarah
Amjad Shraim a,b,c,*, Atef Diab d, Awadh Alsuhaimi a, Esmail Niazy e,
Mohammed Metwally e, Maan Amad f, Salim Sioud f, Abdulilah Dawoud a

a
Chemistry Department, Faculty of Science, Taibah University, Almadinah Almunawarah, Saudi Arabia
b
Toxicological Research and Studies Centre, Taibah University, Almadinah Almunawarah, Saudi Arabia
c
The University of Queensland, National Research Centre for Environmental Toxicology (Entox), Brisbane, Queensland, Australia
d
Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, Taibah University,
Almadinah Almunawarah, Saudi Arabia
e
College of Pharmacy, Taibah University, Almadinah Almunawarah, Saudi Arabia
f
Analytical Core Lab, King Abdullah University of Science and Technology, Thowal, Saudi Arabia

Received 5 September 2012; accepted 15 November 2012

Available online 29 November 2012

KEYWORDS Abstract The chemical pollution of water resources is a major challenge facing the humanity in
Wastewater treatment; this century. Pharmaceuticals and personal care products (PPCPs) are a group of emerging environ-
Antibiotics; mental chemical pollutants distinguished by their bioactivity and high solubility. They may also
Emerging environmental cause health complications to humans and living organisms. Pharmaceuticals enter the environ-
pollutants; ment, mainly via wastewater and can eventually reach the surface and ground water. Despite this,
Frequently dispensed drugs; PPCPs received less attention as environmental pollutants than other chemical pollutants (e.g.
Saudi Arabia; heavy metals and pesticides). The purpose of this work was to investigate the presence of some
Tandem LC–MS
of the most frequently dispensed drugs for the residents of Almadinah Almunawarah, Saudi Arabia
in the municipal wastewater before and after treatment. For this purpose, wastewater samples were
collected biweekly from the city’s sewage treatment plant for a period of 4 months and analyzed the
targeted drugs using tandem LC–MS. Out of the 19 investigated drugs, 5 pharmaceuticals have been
found in concentrations greater than the limit of detection in both the influents and effluents of the
sewage treatment plant. As expected, the concentrations of investigated pharmaceuticals in the
wastewater were found to be low. These drugs and their average concentrations (in ng mL 1) in
the influents were: acetaminophen (38.9), metformin (15.2), norfluoxetine (7.07), atenolol (2.04),

* Corresponding author at: Chemistry Department, Faculty of

Science, Taibah University, P.O. Box 30002, Almadinah Almunawa-
rah, Saudi Arabia. Tel.: +966 556139529; fax: +966 48454770.
E-mail addresses: [email protected], [email protected] (A.
Shraim).
Peer review under responsibility of King Saud University.

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S720 A. Shraim et al.

and cephalexin (1.88). Meanwhile, the effluents contained slightly lower levels (in ng mL 1) than
those of influents: acetaminophen (31.2), metformin (3.19), norfluoxetine (7.25), atenolol (0.545),
and cephalexin (1.53). The results of this study supported by many other investigations indicate
the inefficiency of current conventional wastewater treatment protocols in eliminating such a group
of active and potentially hazardous pollutants from the wastewater.
ª 2012 Production and hosting by Elsevier B.V. on behalf of King Saud University. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

1. Introduction remain bioactive even at extremely low concentrations after

excretion from the body or after disposal to landfills and
Pharmaceuticals and personal care products (PPCPs) are a waters, have unpredictable biochemical interactions when
group of substances that ‘‘refer, in general, to any product mixed together, and may have a tendency to accumulate in
used by individuals for personal health or cosmetic reasons the food chain with negative health impact on aquatic organ-
or used by agribusiness to enhance growth or health of isms and consumers (Escher et al., 2011; Hernando et al.,
livestock’’ (US-EPA). The term PPCPs contain thousands of 2006; Kümmerer, 2008). As a result, pharmaceuticals and their
different chemical compounds, such as prescription and over- metabolites and by-products are of concern for their potential
the-counter therapeutic drugs, veterinary drugs, perfumes, ecological and environmental impacts.
musks, deodorants, shampoos, hair sprays, hair dyes, body lo- Recent literature indicates that the flux of pharmaceuticals
tions, sun-screens, make-up, nail polish, lipsticks, crèmes, diag- from municipal sewage treatment plants (STP) is a consider-
nostic agents, and nutraceuticals. Many of these compounds able source of chemical pollution in surface, ground, marine,
including pharmaceuticals, the focus of this investigation, are and even tap and bottled waters (Chang et al., 2007; Heberer,
bioactive, metabolize partially, and biodegrade slowly (Debska 2002; Khan and Ongerth, 2002; Kolpin et al., 2002; Rosal
et al., 2004; Hernando et al., 2006; Kümmerer, 2008). et al., 2010; Ternes, 1998). For instance, an investigation con-
Little attention has been paid to PPCPs in general and ducted by the U.S. Geological Survey in 1999 to check the
pharmaceuticals in particular as potential environmental pol- occurrence of PPCPs (e.g. sterols, hormones, pharmaceuticals,
lutants when compared to other chemicals pollutants like hea- antibiotics) in surface and ground water has confirmed the
vy metals and pesticides. Interest in pharmaceuticals and their presence of at least one PPCP at low levels in more than two
metabolites and by-products as environmental pollutants has thirds of the samples, with steroids, nonprescription drugs,
only initiated in the 1970s, but it was not until recently when and pesticides being the most frequently detected compounds
scientists actively began to address the impact of such pollu- (Kolpin et al., 2002). Although the concentrations of individ-
tants on the environment and living organisms (Daughton, ual pharmaceuticals reported in investigated water bodies
2002; Debska et al., 2004; Fatta et al., 2007; Heberer, 2002; worldwide are low and may not cause any harm to the human
Hernando et al., 2006; Kümmerer, 2009; Stan and Heberer, health, chronic exposure to a mixture of such compounds may
1997; Zuccato et al., 2006). disturb the balance in the human body and enhance a danger-
Current literature shows that pharmaceuticals are continu- ous resistance to antibiotics and consequently pose a threat to
ously released into the environment in extremely large quanti- the health of living organisms; a task that many scientists are
ties and on a regular basis through different ways like human currently investigating (Escher et al., 2011; Hernando et al.,
activities (e.g. through excretion and bathing and disposal of 2006; Santos et al., 2007; Schriks et al., 2010). Some of the re-
unwanted medications to sewers and trash), wastes from phar- ported potential effects of PPCPs on living organisms were: de-
maceutical industries, residues and wastes from hospitals, use layed development in fish and frogs, delayed metamorphosis in
of illicit and veterinary drugs (especially antibiotics and ste- frogs, increased feminization of fish populations, and a variety
roids), and agribusiness (Bartelt-Hunt et al., 2009; Escher of reactions including altered behavior and reproduction
et al., 2011; Larsson et al., 2007; Ternes, 1998). (Hernando et al., 2006).
Due to the fact that pharmaceuticals generally dissolve eas- The aim of this work was to investigate the occurrence of
ily in aqueous media and do not usually evaporate at normal the most frequently prescribed drugs in the influents and efflu-
temperatures or pressure, they make their way into the soil ents of municipal wastewater of Almadinah Almunawarah.
and aquatic environments via sewage, treated sewage sludge
(biosolids), and irrigation with reclaimed waters 2. Materials and methods
(Cunningham, 2008; Nikolaou et al., 2007). Current research
findings clearly demonstrate that current conventional waste- The list of all pharmaceuticals distributed to the public hospi-
water treatment technologies do not sufficiently remove tals and medical clinics in Almadinah Almunawarah area for
pharmaceuticals and/or their metabolites and degradation the year 2009 was obtained from the Directorate General of
by-products from wastewater, and therefore let them reach Health Affairs of Almadinah Almunawarah. Some of the most
surface, marine, ground, and drinking waters (Benotti and frequently dispensed drugs have been selected to be investi-
Brownawell, 2007; Debska et al., 2004; Joss et al., 2008). gated in this study, based on their quantities and the possibility
Although some pharmaceuticals breakdown or degrade of being detected in wastewater. The targeted drugs and their
upon consumption or release into the environment, most of amounts are presented in Table 1.
them remain unchanged and eventually become persistent in All glass- and plastic-wares used were soaked overnight in
the environment. It is known that most of these chemicals 10% nitric acid, rinsed with distilled water, and finally with
Analysis of some pharmaceuticals in municipal wastewater of Almadinah Almunawarah S721

containing the stock solutions were wrapped with aluminum

Table 1 List of the most frequently dispensed drugs in the
foil and stored refrigerated at 4 C. Working solutions of single
area of Almadinah Almunawarah along with their quantities
analytes as well as mixtures of analytes (10.0 ng mL 1 each)
for the year 1429H (2009).a
were prepared in methanol as required from the stock solu-
Drug’s Drug’s Active tions and stored in a similar way as for the stock solutions.
Group name ingredient (kg) Three types of HPLC mobile phases were prepared: formic
Antibiotics Cephalexin 1,034 acid (A1, 0.3% (v/v) in water); ammonium hydroxide (A2,
Erythromycin ethyl 475 20 mM in water); and acetonitrile: methanol (B, 2:1).
succinate
Antiparasites Metronidazole 2,253 2.2. Samples collection and pre-treatment
Antimicrobials Sulfamethoxazole 1,663
Trimethoprim 333
Anticoagulants Warfarin sodium salt 1.1 Wastewater samples were collected from the only STP in
Antihypertensives Atenolol 269 Almadinah Almunawarah. The current total capacity of this
ACE inhibitors Captopril 120 plant is 300,000 m3 day 1. In addition to domestic sewage,
Hypolipidemic Simvastatin 20 the plant sometimes receives partially-treated industrial and
agents medical wastewater. The plant undertakes tertiary treatment.
Acetaminophen 12,879 Most of the treated wastewater is discharged into the nearby
Non-opioid Ibuprofen 5342
Al-Khlail wadi (valley) and used mainly for irrigation, and
analgesics
about 15% of this water is transported in trucks and used
Diclofenac sodium salt 521
Antiepileptics Carbamazepine 556 for watering of green areas and trees in public parks and streets
Ranitidine 749 of Almadinah Almunawarah.
GI disorders Hyoscine-N-butyl bromide 51 Wastewater samples have been collected biweekly for a per-
Antidiabetics Metformin 6,300 iod of 4 months both from the inlet of the plant after initial
Respiratory drugs Chlorpheniramine malate 431 screening (influents) and from the outlet after chlorination (ter-
Antidepressant Norfluoxetine 2.1 tiary treated, effluents). Composite samples were collected for
a
The most frequently dispensed drug(s) from most of the drug 24 h using a portable water sampler (WS750 dual bottle sam-
classes have been selected. pler from Global Water, CA, USA) at a rate of 100 mL h 1.
The procedure described by Batt et al. (2008) for the anal-
ysis of pharmaceuticals in waste and surface water was
reagent water before use. Glasswares used for the preparation
adopted for this work with modifications. Batt et al. procedure
and storage of drug solutions were rinsed with dimethyldichlo-
was developed and applied for the analysis of pharmaceuticals
rosilane (DMDCS) followed by two toluene rinsings and
in wastewater and surface water samples obtained from New
several methanol washings before use.
Mexico and East Fork River in Cincinnati, Ohio, respectively.
The pH of the samples was measured upon arrival to the lab-
2.1. Chemicals and reagents
oratory and found to be close to neutral values (pH 7.2–7.6)
and therefore the samples were used without any pH adjust-
The following chemicals and reagents were used: reagent water ment. The samples were then filtered using a vacuum filter fun-
(Milli-Q, 18.2 MO cm, Elix10, Millipore, USA), ethylenedi- nel (porosity 25–50 lm, Aldrich). To each 500 mL of filtered
aminetetraacetic acid disodium salt dehydrate (Na2ETDA, samples, a 2 mL solution containing Na2ETDA (5.00 g L 1,
Sigma, ACS reagent, 99.0–101.0%), ascorbic acid (Sigma, ul- used as a metal chelating agent) and ascorbic acid (25.0 mg L 1,
tra grade, >99.0%), formic acid (Fischer Scientific, analytical used to remove any chlorine residues that may be present in
reagent grade, 98%), ammonium hydroxide solution (Sigma– the samples) was added before extraction.
Aldrich, ACS reagent, NH3 content 28–30%), methanol (Sig-
ma–Aldrich, Chromasolv grade, 99.9% min), acetonitrile
(ACN, Fisher Scientific, HPLC Gradient grade), dimethyldi-
chlorosilane (DMDCS, 5% in toluene), and toluene (GC
grade, 99.5%, Panreac, Barcelona-Spain). The drugs of inter- Table 2 Common MS settings.
est were: acetaminophen (98%), carbamazepine, diclofenac Ion source polarity Positive and negative ion modes
sodium salt, erythromycin ethyl succinate, norfluoxetine Capillary voltage 3500 V
(>97%), ranitidine hydrochloride, scopolamine N-butyl Vaporizer temperature 300 C
bromide (hyosine-N-bytyl bromide, >99%), and trimetho- Nebulizer Pressure 45 psi
prim (>98.5%) which were purchased from Sigma–Aldrich, Gas Flow 10 L min 1
ibuprofen (>99%), metronidazole (99.9%), sulfamethoxazole Time Filter True
(99.9%), and warfarin sodium salt (99.9%) from Fluka, and Time Filter Width Wide
atenolol, captopril, cephalexin, chlorpheniramine maleate, MS1 Resolution Wide
MS2 Resolution Wide
metformin hydrochloride (99.8%), and simvastatin (98.3%)
Dwell Time 10 ms
were obtained as a gift from Al-Jazeera Pharmaceutical Indus-
Fragmenter Voltage 135 V
tries, Riyadh, Saudi Arabia. Stock solutions of these drugs Cell Acceleration Voltage 7V
(2000 lg mL 1) were prepared as follows: approx. 20.0 mg Experiment Type: MRM
of each drug was accurately weighed in a glass test tube and MS Run Time 13.0 min (acidics and neutrals)
dissolved in 10 mL water or MeOH depending on analyte sol- and 18.0 min (basics)
ubility. In order to minimize drugs’ degradation, the test tubes
S722 A. Shraim et al.

Table 3 Specific MS settings (all analyzed in + ESI mode, except diclofenac sodium salt and ibuprofen for which –ESI more was
used).
Name RTa Formula Wt Precursor Ion Product Ion 1 Product Ion 2 CEb DTc LODd
Acetaminophen 0.94 151.17 152.2 110.0 93.0 11 10 0.90
Atenolol 0.80 266.34 267.6 145.2 74.2 23 10 0.22
Captopril 0.63 217.29 218.5 70.0 115.5 23 10 6.1
Carbamazepine 5.36 236.27 237.5 194.4 179.3 15 10 0.25
Cephalexin 0.92 365.41 348.9 158.0 174.9 7 10 0.32
Chlorpheniramine maleate 3.85 390.86 275.6 230.4 – 11 10 0.35
Diclofenac sodium salt 3.69 318.13 295.8 250.0 – 4 100 1.33
Erythromycin Ethylsuccinate 1.25 862.1 862.5 387.1 – 15 10 2.1
Ibuprofen 4.36 207.23 207.1 161.0 118.9 2 100 0.91
Hyoscine-N-butylbromide 2.20 440.38 360.1 194.2 – 23 10 1.0
Metformin 0.70 129.16 130.1 60.1 71.1 19 10 3.0
Metronidazole 2.80 171.15 172.2 128.1 82.0 11 10 0.32
Norfluoxetine 3.67 295.3 295.8 278.6 137.8 15 10 1.1
Ranitidine hydrochloride 0.80 350.86 315.8 130.0 127.1 11 10 0.11
Simvastatin 11.94 418.57 419.7 225.4 244.5 5 100 1.0
Sulfamethoxazole 0.69 253.28 254.6 156.0 189.2 11 10 0.21
Trimethoprim 0.90 290.32 291.7 122.9 – 19 10 0.23
Warfarin sodium salt 12.306 308.31 309.8 252.4 164.0 11 10 1.8
a
RT is the retention time (min) of pure standards using A1:B mobile phase except for diclofenac sodium salt and ibuprofen A2:B mobile
phase was used.
b
CE is collision energy (eV).
c
DT is Dwell Time (ms).
d
LOD is the limit of detection in ng mL 1.

Analytes in the samples were extracted at the same day of the second one as ‘‘basics.’’ Reconstituted samples were trans-
collection using Oasis MCX cartridges (mixed mode, 150 mg ferred to glass vials and analyzed by LC–MS. This procedure
from Waters, Milford, MA, USA). The material inside these has been first tried using solutions of mixed standards as
cartridges is made of hydrophobic-lipophilic balanced copoly- described in the section 2.3.2 below.
mer that can retain acidic, basic, and neutral analytes. A drug
behaves as an acid, base, or neutral in solution based on the 2.3. Samples analysis
functional group(s) it processes and on the pH of the solution.
In this context, ‘‘acidic and neutral’’ analytes described in this 2.3.1. UV analyses
work are those pharmaceuticals that are eluted off the SPE In order to identify absorption wavelengths suitable for mon-
Oasis MCX cartridge with ACN, whereas basic analytes are itoring the analytes during the method development, a UV
those that are eluted by ACN in 5% ammonium hydroxide. scan for each analyte was performed using a UV–Vis spec-
The reason for dividing the analytes into 2 groups (‘‘acidic trometer (Jasco, Ubest V-50) as follows: 10.0 mg L 1 solution
and neutrals’’ and basics) was to enhance the sensitivity of of each drug was prepared in the A1-B mobile phase (1:1 ratio)
the MS detector and to avoid any complexity during analysis. and the spectra were taken in the range of 200–400 nm using
Before extraction, each SPE cartridge was conditioned with quartz cells. Same procedure was repeated but using the other
ACN (6 mL) followed by reagent water (6 mL). Samples pre- mobile phase (A2–B, ratio1:1). Blank solutions were also ana-
pared as described above were passed through reconditioned lyzed using both A1–B and A2–B mobile phases in 1:1 ratios.
cartridges at a rate of 3–5 mL min 1 with the aid of a vacuum
pump. Each cartridge was then slowly rinsed with a 2 mL solu- 2.3.2. HPLC-DAD analyses
tion of formic acid (2%) and allowed to dry under vacuum. Method development was undertaken using an HPLC system
Acidic and neutral analytes in each sample were first eluted (1290 series, Agilent Technologies, USA) equipped with a diode
with ACN (2 · 4 mL) into a small glass tube using a vacuum array detector (DAD). For the separation of the analytes, a
manifold (20 positions from Waters, Milford, MA, USA). Ba- SunFire column (C18, 2.1 · 150 mm, 3.5 lm, Waters, Milford,
sic analytes retained on the cartridge material were then eluted MA, USA) preceded by a guard column (SunFire, C18,
by ACN solution (2 · 4 mL) containing ammonium hydroxide 2.1 · 10 mm, 3.5 lm, Waters, Milford, MA, USA) was used
(5%) into a separate glass tube. Each eluate was then concen- at a temperature of 40 C. A gradient elution program was
trated to dryness with the help of a TurboVap LV Concentra- set as follows with a mobile phase flow rate of 0.5 mL min 1.
tion Evaporator Workstation (Caliper Life Sciences, Runcorn, For acidic and neutral analytes, the A2–B mobile phase was
UK) at 40 C under a gentle stream of N2. The contents of the used with an initial mobile phase of 10% solvent B and held
first tube were reconstituted with ACN in water (0.50 mL, for 0.5 min. The ratio of the mobile phase B was then linearly
20 + 80), whereas the eluate in the other tube was reconsti- increased to 40% over 1 min and held for 2.5 min, then to
tuted with MeOH in water (0.50 mL, 20 + 80). The first frac- 90% over 0.5 min and held for 0.5 min. The initial mobile phase
tion is termed from now on as ‘‘acidics and neutrals,’’ whereas composition was restored in 0.5 min and the column was equil-
Analysis of some pharmaceuticals in municipal wastewater of Almadinah Almunawarah S723

Figure 1 Extracted MRM chromatogram (1a), mass spectrum (1b), and product ion spectrum (1c) for acetaminophen.

ibrated for an additional 7.5 min (total run time is 13.0 min). procedure employed for the samples (see Section 2.2). Blank
The DAD wavelength was set at 210 nm (this wavelength was solution has been treated in a similar way. Consequently, a
found to be suitable for all analytes as discussed in Section 20 lL of each of the reconstituted solutions of the two groups
3.1). For the basic analytes, the A1–B mobile phase was used of analytes was injected to the HPLC column. Acidic and neutral
with an initial composition of 10% B and was held for analytes were analyzed using the A2–B mobile phase, whereas
0.5 min. The ratio of the mobile phase B was then linearly in- basic analytes were analyzed using the A1–B mobile phase.
creased to 30% over 0.5 min, then to 35% over 2 min and held
for 7 min, and finally to 90% over 1 min and held for 0.5 min.
The initial mobile phase composition was restored in 0.5 min 2.3.3. LC–MS/MS analyses
and the column was equilibrated for 6.0 min (total run time is Due to the superior sensitivity of MS detection when com-
18.0 min). The following wavelengths have been found to be pared to UV as well as the complexity of the samples matrix,
suitable for detecting basic analytes 225, 230, 240, and MS was used as a detector.
270 nm (refer to Section 3.1 for details). As a mean to identify the molecular ion masses and the
To determine the elution pattern of the analytes, a 20 lL of retention times of the analytes, a 10 lL solution of each ana-
each drug solution (1000.0 lg mL 1) was individually injected lyte (1000.0 lg mL 1) was injected to the LC–MS system (Agi-
to the HPLC column using the two HPLC protocols described lent 1290 UHPLC and 6460 MS/MS series with Jet Steam ESI
above. Following this, the two groups of analytes eluted from source) using a mobile phase flow rate of 0.5 mL min 1. Then,
the SPE as acidics and neutrals (group 1) or as basics (group a product ion scan employing the multiple monitoring reaction
2) have been identified as follows: a mixed standard solution mode (MRM) was performed to collect data for suitable prod-
containing all analytes (10 mL of 1000.0 lg mL 1) has been ex- uct ions. The two most intense MRM transitions were then se-
tracted, eluted, evaporated, and reconstituted using the same lected for all analytes except for chlorpheniramine maleate,
S724 A. Shraim et al.

Figure 2 Extracted MRM chromatogram (2a), mass spectrum (2b), and product ion spectrum (2c) for atenolol.

diclofenac sodium salt, erythromycin ethylsuccinate, hyoscine- more than the LOD and the ratio of the ions is within 30% of
n-butylbromide, and trimethoprim, for which only one prod- the anticipated ratio.
uct ion was present. The MRM transitions were optimized The limit of detection (LOD) for each analyte was deter-
using different collision energies. The utilized common MS set- mined using 5–7 replicate injections of a reagent blank and
tings are listed in Table 2, whereas the specific settings were was calculated as the average concentration measured for the
shown next to each analyte in Table 3. After establishing the blank multiplied by 3 times its standard deviation.
MRM, the two groups of analytes (extracted by the cartridges
as described at the end of Section 2.3.3) have also been injected
to the LC–MS system to confirm the results obtained by 3. Results and discussion
HPLC-DAD. Finally, calibration standard solutions and trea-
ted samples solutions were injected to the LC–MS system. For 3.1. UV analysis
quantification of the analytes, a 4-point calibration curve for
each analyte was constructed at concentrations of 0.000, According to the UV spectra of all analytes (not shown here)
100.00, 500.00, and 1000.0 lg mL 1. The R2-value for the an optimal wavelength of 210–215 nm can be used for all ana-
curves was better than 0.998. For LC–MS analysis, same lytes when using the A2–B (1:1) mobile phase (210 nm was
chromatographic conditions (i.e. column, mobile phases, and used). On the other hand, the use of the A1–B (1:1) mobile
gradient elution programs) used for HPLC-DAD analyses phase resulted in a different spectral pattern and a different
were employed. For detection of the analytes both the reten- optimal wavelength was obtained for most of the analytes.
tion time and product ion ratios were used. Analytes were pos- Additionally the following compounds showed no absorption
itively identified if both product ions are present in abundance signals in the investigated wavelength range: captopril, eryth-
Analysis of some pharmaceuticals in municipal wastewater of Almadinah Almunawarah S725

Cephalexin (3a)

Cephalexin (3b)

Cephalexin
(3c)

Figure 3 Extracted MRM chromatogram (3a), mass spectrum (3b), and product ion spectrum (3c) for cephalexin.

romycin ethyl succinate, and hyosine-N-butyl bromide. As a Supelco C8, Supelco C18, Varian Focus, and Merck
consequence multiple wavelengths have been used to monitor LiChrolut-EN. The Oasis MCX, which was employed in this
the analytes: 225, 230, 240, and 270 nm; each of them has been work, has been shown to provide high overall recoveries. (Batt
used to monitor a sub group of the analytes. et al., 2008; Lacey et al., 2008).
Extracted MRM chromatogram, mass spectrum, and prod-
3.2. HPLC-DAD analysis uct ion spectrum for each analyte that has been detected in the
wastewater in concentrations greater than the LOD are shown
Experiments conducted as described in Section 2.2 above in Figs. 1–5. Out of the 19 targeted drugs, 5 pharmaceuticals
showed that the analytes eluted as basics were: atenolol, rani- have been found both in raw (influents) and treated (effluents)
tidine, simvastatin, and trimethoprim. In contrast, erythromy- wastewater samples. The concentrations of these pharmaceuti-
cin ethyl succinate showed no HPLC-DAD signal, but cals are shown in Tables 4 and 5. Although most pharmaceu-
confirmed by LC–MS to elute in the basics group. The rest ticals have high solubility in water and hence remain soluble in
of the analytes was eluted in the acidics and neutrals fraction. the aqueous phase, some drugs have lower solubility and re-
main insoluble as the solid material in wastewater. Since the
3.3. LC–MS/MS Analysis samples were filtered before extraction, the reported concen-
trations in this work represent only the water-soluble fraction
Several solid phase extraction cartridges were used in the liter- of the analytes.
ature for the extraction of pharmaceuticals from wastewater The detected drugs have been found in most of the samples as
including Oasis HLB, Oasis MCX, Oasis WCX, Strata-X, shown in Tables 4 and 5. As expected, the concentrations of indi-
S726 A. Shraim et al.

Figure 4 Extracted MRM chromatogram (4a), mass spectrum (4b), and product ion spectrum (4c) for metformin.

vidual pharmaceuticals in the wastewater influents were found to around 70 pharmaceuticals and PPCPs in influents of
be low with an average concentration of 2.04–38.9 ng mL 1. municipal wastewater with some compounds in the ng mL 1
The highest detected concentration was for acetaminophen concentration range (e.g. paraxanthine, caffeine, and acetami-
(99.6 ng mL 1), followed by metformin (31.2 ng mL 1), nor- nophen), whereas the rest were in the ng L 1 range (Rosal
fluoxetine (10.4 ng mL 1), atenolol (4.03 ng mL 1), and cepha- et al., 2010). In another study by Gracia-Lor et al., 13 out of
lexin (3.23 ng mL 1). Interestingly, the amounts of these 20 investigated drugs were detected in the influents of urban
pharmaceuticals in the treated wastewater (effluents) did not dif- wastewater samples with salicylic acid having the highest
fer much from those found in the raw wastewater (influents), with concentration (276.7 ng mL 1) (Gracia-Lor et al., 2010).
a concentration range of 0.545–31.2 ng mL 1. The highest con- On the other hand, effluents of urban wastewater and
centration detected was for acetaminophen (90.5 ng mL 1), fol- receiving waters were reported also to contain many pharma-
lowed by norfluoxetine (11.7 ng mL 1), cephalexin (2.83 ng mL 1), ceuticals at low concentrations. (Bartelt-Hunt et al., 2009;
metformin (4.51 ng mL 1), and atenolol (2.01 ng mL 1). Gracia-Lor et al., 2010; Joss et al., 2008; Lacey et al., 2008;
The findings of this study is consistent with those of many Santos et al., 2007; Soliman et al., 2007; Spongberg and Witter,
reports in the literature, where low concentrations of pharma- 2008; Ternes, 1998; Zhou et al., 2009) Clearly, this indicates
ceuticals have been found in municipal wastewater that most of the current wastewater treatment practices are
(Bartelt-Hunt et al., 2009; Batt et al., 2008; Gracia-Lor et al., inefficient in completely removing such contaminants. For in-
2010; Miège et al., 2009; Radjenovic et al., 2009; Rosal et al., stance, 5 pharmaceuticals (i.e. propranolol, sulfamethoxazole,
2010). For example, Rosal et al. reported the occurrence of carbamazepine, indomethacine and diclofenac) were found in
Analysis of some pharmaceuticals in municipal wastewater of Almadinah Almunawarah S727

Norfluoxetine (5a)

Norfluoxetine (5b)

Norfluoxetine
(5c)

Figure 5 Extracted MRM chromatogram (5a), mass spectrum (5b), and product ion spectrum (5c) for norfluoxetine.

all wastewater and the receiving surface water samples in sludge used in the plant, but the main factor is reported to be
England with carbamazepine having the highest levels the physico-chemical nature of most pharmaceuticals, which is
(2.336 ng mL 1). The reported removal efficiencies for these the acidity and high solubility in water with very low solid–
compounds from the wastewater were in the range of 43– liquid partition. These factors, especially the last one lead to
92%.(Zhou et al., 2009) In another study, 5 out of 6 drugs (dic- a very poor sorption of these compounds onto sludge and
lofenac, ibuprofen, ketoprofen, naproxen, carbamazepine, and hence leaving them soluble in the aqueous phase (Gracia-Lor
caffeine) have been detected in both influent and effluent sam- et al., 2012; Verlicchi et al., 2012).
ples from 4 STPs in Seville–Spain in the ng mL 1 concentra- It is also worth noting here that some of the pharmaceuticals
tion range. The reported removal rates for these drugs were that were detected in the wastewater were also found in the
between 6% and 98%. (Santos et al., 2007) Similarly, 5 out sludge of the wastewater treatment plans. This is due to the
of 10 pharmaceuticals detected in the influents of 3 STPs in low solubility of such drugs and therefore they remain insoluble
Duplin-Ireland have been found in the wastewater effluents and appear in the sludge (Gao et al., 2012; Jelic et al., 2011).
with concentrations of <1 ng mL 1 for most samples (Lacey When considering a single pharmaceutical at low concen-
et al., 2008). trations such as those reported in this investigation and other
The removal efficiency of pharmaceuticals from wastewater work, it may be assumed that not many health risks can be
is actually dependent on several factors including the climatic associated with long term exposure to such a drug. But the
conditions, the type of wastewater treatment and its opera- health risks associated with exposure to a large number of
tional conditions (e.g. temperature, redox conditions, solids pharmaceuticals, their metabolites, and transformation prod-
and hydraulic retention time) as well as the age of the activated ucts, even at low concentrations, cannot be ignored.
S728 A. Shraim et al.

Table 4 Concentrations of pharmaceuticals (ng mL 1) in raw wastewater samples.

Sample IDa Metformin Atenolol Cephalexin Acetaminophen Norfluoxetine
1R 9.01 2.04 2.52 99.6 10.4
2R 16.5 2.02 2.04 58.7 9.11
3R 17.0 4.03 <MDL 40.5 8.43
4R 4.02 1.04 2.01 3.61 10.4
5R 13.0 2.99 3.23 29.0 7.03
6R 25.1 2.02 1.48 36.7 7.64
7R 31.2 1.97 2.09 5.51 2.48
8R 5.93 <MDL 1.37 37.7 <MDL
Range 4.02–31.2 <MDL - 4.03 <MDL - 3.23 3.61–99.6 <MDL - 10.4
Median 14.8 2.02 2.03 37.2 8.04
Average 15.2 2.04 1.88 38.9 7.07
a
R means raw wastewater (influents).

Table 5 Concentrations of pharmaceuticals (ng mL 1) in treated wastewater samples.

a
Sample ID Metformin Atenolol Cephalexin Acetaminophen Norfluoxetine
1T <LOD 2.01 2.21 16.1 11.7
2T <LOD <LOD 1.68 <LOD 9.81
3T <LOD 1.03 <LOD <LOD 9.72
4T <LOD <LOD 1.27 48.8 8.17
5T <LOD <LOD 2.83 <LOD 9.57
6T <LOD <LOD <LOD 90.5 <LOD
7T <LOD <LOD 1.74 44.8 6.83
8T 4.51 <LOD 1.88 46.7 <LOD

Range <LOD - 4.51 <LOD - 2.01 <LOD - 2.83 <LOD – 90.5 <LOD – 11.7
Median <LOD <LOD 1.71 30.5 8.87
Average 3.19 0.545 1.53 31.2 7.25
a
T means treated wastewater (effluents).

4. Conclusions like to thank the Directorate General of Health Affairs of

Almadinah Almunawarah for providing the list of pharmaceu-
ticals dispensed to the public hospitals and health clinics in
Out of the 19 pharmaceuticals investigated in this study, 5
Almadinah Almunawarah. Special thanks to Mr. Mohammad
drugs have been found both in the influents and effluents of
Ghazal and Mr. Abdallah Mahrous from the Wastewater Treat-
the STP. The concentrations of these drugs in both types were
ment Plant – Almadinah Almunawarah for providing access to
in the lower ng mL 1 (influents range 2.04–38.9, max. 99.6;
the Plant. The authors are also grateful to Dr. Nabil Fayad from
effluents range 0.545–31.2, max. 90.5) with effluents having
King Abdallah University for Science and Technology and to
slightly lower concentrations than the influents in most cases.
Mr. Ramesh Muthukumarasamy, Mr. Mahesh D Ganapathy,
The results of this investigation, supported by a similar
Mr. Sampath Alwar, and Mr. Ahmad Bakr from Integrated
work in the literature, indicate that many drugs (including
Modern Scientific Supplies – Saudi Arabia for providing access
their metabolites and transformation products) are not effi-
to their facilities and participating in samples analysis.
ciently eliminated during the wastewater treatment processes
(sometimes tertiary treatment, as in this study). This may sug-
gest that conventional wastewater treatment technologies are References
inefficient in completely removing such compounds and as a
consequence leaving the way open for such bioactive com- Bartelt-Hunt, S.L., Snow, D.D., Damon, T., Shockley, J., Hoagland,
pounds to enter the aquatic environment and eventually pol- K., 2009. The occurrence of illicit and therapeutic pharmaceuticals
lute the drinking water supplies and pose health risks to in wastewater effluent and surface waters in Nebraska. Environ.
humans and other living organisms. Pollut. 157 (3), 786–791.
Although, the levels of detected pharmaceuticals in the trea- Batt, A.L., Kostich, M.S., Lazorchak, J.M., 2008. Analysis of
ted water are quite low, the health risks associated with long ecologically relevant pharmaceuticals in wastewater and surface
term exposure to a large number of pharmaceuticals have to water using selective solid-phase extraction and UPLC–MS/MS.
Anal. Chem. 80 (13), 5021–5030.
be kept in mind.
Benotti, M.J., Brownawell, B.J., 2007. Distributions of pharmaceuti-
cals in an urban estuary during both dry- and wet-weather
Acknowledgements conditions. Environ. Sci. Technol. 41 (16), 5795–5802.
Chang, H., Hu, J.Y., Shao, B., 2007. Occurrence of natural and
This research was funded by the Deanship of Scientific Research synthetic glucocorticoids in sewage treatment plants and receiving
– Taibah University (Project No. 429-233). The authors would river waters. Environ. Sci. Technol. 41 (10), 3462–3468.
Analysis of some pharmaceuticals in municipal wastewater of Almadinah Almunawarah S729

Cunningham, V.L., 2008. Special characteristics of pharmaceuticals ceutical compounds in wastewater treatment plant influent and
related to environmental fate. In: Kümmerer, K. (Ed.), Pharma- effluent samples. Talanta 75 (4), 1089–1097.
ceuticals in the Environment: Sources, Fate, Effects and Risks. Larsson, D.G.J., de Pedro, C., Paxeus, N., 2007. Effluent from drug
Springer, Berlin, pp. 23–34. manufactures contains extremely high levels of pharmaceuticals. J.
Daughton, C.G., 2002. Environmental stewardship and drugs as Hazard. Mater. 148 (3), 751–755.
pollutants. The Lancet 360, 1035–1036. Miège, C., Choubert, J.M., Ribeiro, L., Eusèbe, M., Coquery, M.,
Debska, J., Kot-Wasik, A., Namiesnik, J., 2004. Fate and analysis of 2009. Fate of pharmaceuticals and personal care products in
pharmaceutical residues in the aquatic environment. Crit. Rev. wastewater treatment plants – conception of a database and first
Anal. Chem. 34 (1), 51–67. results. Environ. Pollut. 157 (5), 1721–1726.
Escher, B.I., Baumgartner, R., Koller, M., Treyer, K., Lienert, J., Nikolaou, A., Meric, S., Fatta, D., 2007. Occurrence patterns of
McArdell, C.S., 2011. Environmental toxicology and risk assess- pharmaceuticals in water and wastewater environments. Anal.
ment of pharmaceuticals from hospital wastewater. Water Res. 45 Bioanal. Chem. 387 (4), 1225–1234.
(1), 75–92. Radjenovic, J., Petrovic, M., Barcel, D., 2009. Fate and distribution of
Fatta, D., Nikolaou, A., Achilleos, A., Meric, S., 2007. Analytical pharmaceuticals in wastewater and sewage sludge of the conven-
methods for tracing pharmaceutical residues in water and waste- tional activated sludge (CAS) and advanced membrane bioreactor
water. Trends Anal. Chem. 26 (6), 515–533. (MBR) treatment. Water Res. 43 (3), 831–841.
Gao, P., Ding, Y., Li, H., Xagoraraki, I., 2012. Occurrence of Rosal, R., Rodrguez, A., Perdign-Meln, J.A., Petre, A., Garcáa-Calvo,
pharmaceuticals in a municipal wastewater treatment plant: mass E., Gmez, M.J., Agüera, A., Fernández-Alba, A.R., 2010. Occur-
balance and removal processes. Chemosphere 88 (1), 17–24. rence of emerging pollutants in urban wastewater and their removal
Gracia-Lor, E., Sancho, J.V., Hernández, J.V., 2010. Simultaneous through biological treatment followed by ozonation. Water Res. 44
determination of acidic, neutral and basic pharmaceuticals in urban (2), 578–588.
wastewater by ultra high-pressure liquid chromatography–tandem Santos, J.L., Aparicio, I., Alonso, E., 2007. Occurrence and risk
mass spectrometry. J. Chromatogr. A 1217 (5), 622–632. assessment of pharmaceutically active compounds in wastewater
Gracia-Lor, E., Sancho, J.V., Serrano, R., Hernández, F., 2012. treatment plants. A case study: Seville city (Spain). Environ. Int. 33
Occurrence and removal of pharmaceuticals in wastewater treat- (4), 596–601.
ment plants at the Spanish Mediterranean area of Valencia. Schriks, M., Heringa, M.B., van der Kooi, M.M.E., de Voogt, P., van
Chemosphere 87 (5), 453–462. Wezel, A.P., 2010. Toxicological relevance of emerging contami-
Heberer, T., 2002. Tracking persistent pharmaceutical residues from nants for drinking water quality. Water Res. 44 (2), 461–476.
municipal sewage to drinking water. J. Hydrol. 266 (3–4), 175–189. Soliman, M.A., Pedersen, J.A., Park, H., Castaneda-Jimenez, A.,
Hernando, M.D., Mezcua, M., Fernandez-Alba, A.R., Barcelo, D., Stenstrom, M.K., Suffet, I.H., 2007. Human pharmaceuticals,
2006. Environmental risk assessment of pharmaceutical residues in antioxidants, and plasticizers in wastewater treatment plant and
wastewater effluents, surface waters and sediments. Talanta 69, water reclamation plant effluents. Water Environ. Res. 79 (2), 156–
334–342. 167.
Jelic, A., Gros, M., Ginebreda, A., Cespedes-Sánchez, R., Ventura, F., Spongberg, A.L., Witter, J.D., 2008. Pharmaceutical compounds in the
Petrovic, M., Barcelo, D., 2011. Occurrence, partition and removal wastewater process stream in Northwest Ohio. Sci. Total Environ.
of pharmaceuticals in sewage water and sludge during wastewater 397 (1–3), 148–157.
treatment. Water Res. 45 (3), 1165–1176. Stan, H.J., Heberer, T., 1997. Pharmaceuticals in the aquatic
Joss, A., Siegrist, H., Ternes, T.A., 2008. Are we about to upgrade environment. Analusis 25 (7), M20–M23.
wastewater treatment for removing organic micropollutants? Water Ternes, T.A., 1998. Occurrence of drugs in German sewage treatment
Sci. Technol. 57 (2), 251–255. plants and rivers. Water Res. 32 (11), 3245–3260.
Khan, S.J., Ongerth, J.E., 2002. Estimation of pharmaceutical residues US-EPA. Pharmaceuticals and Personal Care Products as Pollutants.
in primary and secondary sewage sludge based on quantities of use Available from: <http://www.epa.gov/ppcp/> (last updated 29/
and fugacity modelling. Water Sci. Technol. 46 (3), 105–113. 02/2012, accessed on 04/12/2012).
Kolpin, D.W., Furlong, E.T., Meyer, M.T., Thurman, E.M., Zaugg, Verlicchi, P., Al Aukidy, M., Zambello, E., 2012. Occurrence of
S.D., Barber, L.B., Buxton, H.T., 2002. Pharmaceuticals, hor- pharmaceutical compounds in urban wastewater: removal, mass
mones, and other organic wastewater contaminants in U.S. load and environmental risk after a secondary treatment – A
streams, 1999–2000: a national reconnaissance. Environ. Sci. review. Sci. Total Environ. 429, 123–155.
Technol. 36 (6), 1202–1211. Zhou, J.L., Zhang, Z.L., Banks, E., Grover, D., Jiang, J.Q., 2009.
Kümmerer, K., 2008. Pharmaceuticals in the environment – A brief Pharmaceutical residues in wastewater treatment works effluents
summary. In: Kümmerer, K. (Ed.), Pharmaceuticals in the and their impact on receiving river water. J. Hazard. Mater. 166 (2–
Environment: Sources, Fate, Effects and Risks. Springer, Berlin, 3), 655–661.
pp. 3–22. Zuccato, E., Castiglioni, S., Fanelli, R., Reitano, G., Bagnati, R.,
Kümmerer, K., 2009. Antibiotics in the aquatic environment – A Chiabrando, C., Pomati, F., Rossetti, C., Calamari, D., 2006.
review – Part I. Chemosphere 75 (4), 417–434. Pharmaceuticals in the environment in Italy: causes, occurrence,
Lacey, C., McMahon, G., Bones, J., Barron, L., Morrissey, A., Tobin, effects and control. Environ. Sci. Pollut. Res. 13 (1), 15–21.
J.M., 2008. An LC–MS method for the determination of pharma-