Received: 25 September 2018 Revised: 23 October 2018 Accepted: 16 November 2018

DOI: 10.1002/jssc.201801002

RESEARCH ARTICLE

A rapid and sensitive reversed phase-HPLC method for

simultaneous determination of ibuprofen and paracetamol in
drug samples and their behaviors in simulated gastric conditions

Tülay Borahan1 Tuğçe Unutkan2 Ayşe Şahin3 Sezgin Bakırdere1

1 Department of Chemistry, Yıldız Technical

University, İstanbul, Turkey
2 Department of Chemical Engineering, Yıldız
Technical University, İstanbul, Turkey
3 Department of Pediatric, Sisli Hamidiye Etfal
Training and Research Hospital, İstanbul,
Turkey
Correspondence
Sezgin Bakırdere, Yıldız Technical University,
Department of Chemistry, 34349 İstanbul,
Turkey
Email: bsezgin23@yahoo.com
Paracetamol is a widely used drug for fever and pain relief. Ibuprofen is a common
nonsteroidal anti-inflammatory drug. In this study, a sensitive and accurate reversed
phase high performance liquid chromatography method was developed for the simul-
taneous determination of ibuprofen and paracetamol. The chromatographic separation
was achieved on a Phenomenex C18 (250 mm, 4.6 mm, 5 μm) column. Fifty milli
molar phosphate buffer (pH 7.5) and methanol were used as mobile phase in a gra-
dient elution mode. The retention times of paracetamol and ibuprofen were 5.7 and
10.4 min, respectively. The linearity of the developed method was established in the
range of 0.25 – 250 mg/L with a correlation coefficient of 0.9998 for both analytes. The
limit of detection/quantification values were found to be 0.06/0.19 and 0.08/0.26 mg/L
for ibuprofen and paracetamol, respectively. The method was successfully applied in
drug samples in the form of tablets and suspensions. The calculated concentrations
matched with the claimed values on their prospectuses. The drug samples were stud-
ied under simulated gastric conditions to determine the behaviors of the analytes in
the human body. The obtained results showed no change in the retention time of the
analyte peak shapes throughout the 210 minutes.

KEYWORDS
high-performance liquid chromatography, ibuprofen, paracetamol, simulated gastric juice

1 I N T RO D U C T I O N cramps, headaches, arthritis, and other related pains [10]. The

Paracetamol chemically referred to as N-(4-hydroxyphenyl)
acetamide is the most common drug used all over the world for
its analgesic and antipyretic properties [1,2]. Due to its minor
side effects, it is conveniently used by both young and elderly
people for the relief of minor aches, pain, and fever [3,4]. The
therapeutic dose of paracetamol does not cause any negative
side effects for human health; however, overdose consump-
tion causes damages to the liver and kidneys [5,6]. The drug
is partially soluble in cold water, more soluble in hot water
and alcohols [7]. Ibuprofen known as 2-(4-isobutylphenyl)
propionic acid is a ubiquitous nonsteroidal anti-inflammatory
drug with analgesic and anti-inflammatory properties [8,9]. It
is prescribed for the treatment of fever symptoms, menstrual
drug is available as tablets or capsules ranging in dosage from
400 to 600 mg. It is regarded to be safe for human health
depending on the dose consumed [11]. Nurofen, Advil, and
Motrin are the popular formulations of ibuprofen that are sold
on the market [12]. This chemical is an acidic compound hav-
ing pKa ≈ 4.4 and has poor solubility in water with high mem-
brane permeability [13]. Its solubility in solution is pH depen-
dent due to its ionization property [14].
Several spectrophotometric [15,16], chromatographic [17,
18], and titrimetric [19,20] methods have been reported for
the individual determination of ibuprofen and paracetamol.
HPLC method for ibuprofen tablets and column chromatog-
raphy followed by spectrophotometry for paracetamol tablets
are recommended by the United States Pharmacopeia [21].

J Sep Sci 2018;1–6. www.jss-journal.com © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 1
2 BORAHAN ET AL.

There are limited reported methods in the literature for simul- TABLE 1 Gradient elution program for ibuprofen and
taneous determination in combined formulations [22]. One paracetamol
of these methods is RP-HPLC assay method for paraceta- Time, min Buffer, %v/v Methanol, %v/v
mol, ibuprofen, and chlorzoxazone tablets [23]. Another study 0.01 80 20
reports the determination of ibuprofen and paracetamol by 2.0 80 20
HPLC [24]. Additional reported study in the literature is 9.0 5 95
the simultaneous determination of caffeine, paracetamol, and 12 5 95
ibuprofen in pharmaceutical formulations by HPLC with UV
14 80 20
detection and by capillary electrophoresis with conductiv-
15 80 20
ity detection [25]. HPLC is mostly preferred over gas chro-
matography in pharmaceutical analysis because it is appli-
cable to a wider range of compounds [26]. However, HPLC
takes time for stabilization and optimization of parameters
such as mobile phase and flow rate. In addition to this, it
requires considerable amounts of organic solvents for sample Ultrapure deionized water was obtained from a Milli-Q® Ref-
elution [27,28]. erence System.
The main aim of this study was to develop a sensitive RP-
HPLC method for accurate determination of ibuprofen and
paracetamol in simulated gastric fluid and commercial drugs. 2.3 Chromatographic conditions
The study was also aimed at developing a simple method Chromatographic separation was achieved on a Phenomenex
appropriate for routine analysis of the analytes, and examining C18 (250 mm, 4.6 mm, 5μm) column in a gradient elu-
the behavior of the analytes when ingested into the stomach. tion mode. Fifty milli molar phosphate buffer (pH 7.5)
and methanol were used as mobile phase at a flow rate of
1.0 mL/min. The injection volume was 20.0 μL and the elu-
ents were monitored at 220 nm with the UV detector. The total
2 M AT E R I A L S A N D M E T H O D S chromatographic run period was 17.0 min. The gradient elu-
tion program employed is shown in Table 1.
2.1 Instrumentation
A Shimadzu HPLC (CTO-10AS VP oven, SIL-20A HT auto 2.4 Procedure for commercial drugs
sampler) coupled with a UV detector was employed for the
Tablet samples of each drug were crushed into fine power
simultaneous determination of ibuprofen and paracetamol.
and dissolved with methanol in 100 mL volumetric flasks and
Detection was performed by the SPD-20A Model UV detec-
mixed for 1.0 h on a magnetic stirrer. 5.0 mL of each sus-
tor at 220 nm wavelength. A Phenomenex C18 (250 mm,
pension sample was added to 100 mL volumetric flasks and
4.6 mm, 5μm) column was used for chromatographic sepa-
diluted with methanol, and mixed for 1.0 h on the magnetic
ration of paracetamol and ibuprofen. pH measurements were
stirrer. The solutions were filtered through 0.45 μm syringe
made using a Hanna HI 2211 pH/ORP meter. All solvents
filters and the concentration of the analytes adjusted in accor-
were mixed on an IKA C-MAG HS 7 brand magnetic stirrer.
dance with their linear calibration range. Analyte determina-
tion was performed with the HPLC-UV system under the opti-
mum conditions.
2.2 Reagents and chemicals
Paracetamol and ibuprofen working standards were obtained
from a pharmaceutical company in İstanbul, Turkey. A mixed
2.5 Preparation of simulated gastric juice
standard stock solution (2000 mg/L) of paracetamol and
solution
ibuprofen was prepared by accurately weighing 200 mg of Simulated gastric juice was prepared in a 50 mL volumetric
each analyte and dissolving in a 100 mL volumetric flask with flask by adding 0.10 g NaCl, 0.35 mL HCl (36–37%), and
methanol. Working and calibration standard solutions were 0.16 g pepsin [27]. Before topping up to the 50 mL mark with
prepared by diluting appropriate aliquots from the 2000 mg/L deionized water, the solution was spiked to a final concentra-
mix standard solution. Reagents used for the preparation tion of 100 mg/L. A sample was immediately taken from the
of mobile phase included methanol (>99%) and KH2 PO4 spiked simulated gastric juice and the remaining maintained
(>99%). Pepsin, sodium chloride and hydrochloric acid (36– at 37◦ C in a water bath. Samples were taken from the spiked
37%) were used for simulated gastric fluid studies. All ana- simulated gastric juice and analyzed after every half-hour for
lytical reagents were purchased from Merck (Germany). a period of 210 min.
BORAHAN ET AL.
TABLE 2 Optimum parameters of the HPLC-UV system
Parameter Optimized Value
Mobile Phase Gradient Elution (Table 1)
Buffer Solution 50 mM phosphate buffer
pH 7.5
Flow Rate, mL/min 1.0
Wavelength, nm 220
3 RESULT A N D D I SC U SSI O N
All the system parameters and mobile phase composition were
optimized in order to obtain the most favorable experimental
outcomes. Column, mobile phase (pH, component, gradient
elution) and oven temperature were optimized as shown in
Table 2. The final mobile phase composition and flow rate
allowed separation of ibuprofen (10.4 min) and paracetamol
(5.7 min) in 17 min analysis period (Figure 1).
At the beginning of the optimization, system parameters
were chosen according to the evaluated results in the lit-
erature. Phosphate buffer, methanol (20%) and phosphate
buffer (80%) for mobile phase, 1.0 mL/min flow rate, 210 nm
for wavelength were chosen as the initial system parame-
ters. Fifty milligrams per liter of mix working standard solu-
tion of the analytes was used during each optimization step.
Optimization studies started with changing pH values by
using acidic pH values (3.0 and 5.0) and basic pH values
(7.5 and 10) while keeping other variables constant (mobile
phase composition, flow rate, wavelength, and buffer solu-
tion). Peaks of the analytes were not symmetric and sharp
at pH 3 and 5. Better shapes of the peaks were obtained at
basic medium (pH 7.5) than the other examined pH values.
Therefore, buffer solutions were prepared at basic pH values
(7.5, 8.5, 9.5, and 10.8). By using initial parameters, sharper
and less broad signals were observed at pH 7.5 and the peaks
were fully resolved when compared to the other studied pH
values. Methanol and acetonitrile were studied as organic sol-
vents. Analyte peaks were asymmetric when acetonitrile was
FIGURE 1 Chromatograms of 250 mg/L of ibuprofen and paracetamol
3
given to the system while the peaks were more stable and
symmetric with methanol as the organic solvent. The peak
of paracetamol and the dead time were close when the ini-
tial mobile phase composition (methanol 20% and phosphate
buffer 80%) was examined. Hence, the gradient elution pro-
gram was set as shown in Table 1 in order to obtain the
best resolution. 0.80, 1, and 1.2 mL/min were tried for the
flow rate optimization. 1 mL/min was chosen for giving the
best shape of the peaks. 200, 210, and 220 nm were investi-
gated as wavelengths. The highest peak areas were obtained at
220 nm.
3.1 Analytical performance of ibuprofen and
paracetamol
Calibration curves were constructed using the average peak
area of triplicate measurements recorded for mixed aqueous
standard solutions prepared in the range of 0.25–250 mg/L
(0.25, 0.50, 1, 2.5, 5, 25, 50, 100, and 250 mg/L). Both ana-
lytes exhibited broad linear dynamic range with a correlation
coefficient of 0.9998. The LOD and LOQ values were calcu-
lated with six replicate measurements of the lowest analyte
concentrations in the linear calibration plots using the equa-
tions below.
LOD = 3
(1)
× Standard Deviation (lowest concentration)∕Slope
LOQ = 10
(2)
× Standard Deviation (lowest concentration) ∕Slope
LOD/LOQ values were found to be 0.06/0.19 and
0.08/0.26 mg/L for ibuprofen and paracetamol, respectively.
The %RSD obtained for both analytes were found to be lower
than 10%, indicating high instrumental precision.
4 BORAHAN ET AL.

FIGURE 2 Chromatogram of Brand A suspension sample

3.2 Determination of ibuprofen and 7.5% more paracetamol while Brand A as suspension includes
paracetamol in drug samples 23% higher amount in ibuprofen.

In this study, quantitative determination of the analytes was

performed for tablet and suspension drug samples to deter-
mine whether the amounts of ibuprofen and paracetamol in
3.3 Stability of ibuprofen and paracetamol in
drug samples match the values stated on their prospectuses.
simulated gastric conditions
It is stated that 5 mL suspension of Brand A drug con- In this part of the study, the behavior of ibuprofen and parac-
tains 100 mg ibuprofen while 5 mL suspension of Brand B etamol under simulated gastric conditions were tried to be
drug contains 120 mg paracetamol. The concentrations of figured out. Vials containing simulated gastric juice spiked
the analytes in suspension samples for Brand A and Brand with mixed standards of the analytes to a final concentration
B were diluted to 60 and 40 mg/L, respectively to fall into of 100 mg/L were maintained at 37◦ C in a water bath. Starting
the linear calibration range of the standard samples. The chro- from 0.0 min, samples were taken from the water bath setup
matograms obtained for the samples showed peaks of ibupro- and analyzed under the optimum conditions of the HPLC-UV
fen and paracetamol that matched the retention times obtained system every 30 min until 210 min. It was observed that there
using standard solutions. Figure 2 shows the chromatogram of was no change in the retention times of ibuprofen and parac-
Brand A suspension sample. Tablets of Brand C and Brand D etamol throughout the 210 min period. In order to ensure that
as stated on the labels contain 600 mg ibuprofen and 500 mg there were no matrix effects, spiking experiments were per-
paracetamol per tablet, respectively. The concentrations of formed after samples were taken from the simulated gastric
ibuprofen and paracetamol in tablet samples for Brand C and fluid. The chromatogram obtained showed that there was no
Brand D were prepared within their linear calibration range shift in the retention time of the analyte peaks.
by diluting to 50 and 60 mg/L, respectively. The concen-
tration of active components of the drugs (Table 3) were
determined using their integrated peak area values against
calibration standards. The results obtained were within 4 CONC LU D I NG R E M A R K S
acceptable limits of the amounts stated for each active drug
component. It is clear in Table 3 that there is a problem in A reliable, sensitive, and accurate HPLC method for the
the statements of the Brand A and D. Brand D contains about simultaneous determination of ibuprofen and paracetamol
was developed in this study. Under the optimum conditions,
TABLE 3 HPLC analysis results of different types of drug
the analytical performance of the method was determined
samples for both analytes. The analytes showed good linearity over a
broad calibration range and the other figures of merit were
Drug Prospectus Obtained values,
Sample Drug Form values, mg mg
satisfactory. The method was applied to commercial drugs
including two suspension and two tablet formulations. The
Brand Aa Suspension 100 122.8 ± 2.4
concentrations of the analytes determined in the drug sam-
Brand Ba Suspension 120 120.9 ± 0.3
ples with the optimum method confirmed to the stated value
Brand Ca Tablet 600 594.3 ± 6.6
on the prospectuses. Additionally, stability test of ibuprofen
Brand Da Tablet 500 537.8 ± 0.7
and paracetamol under simulated gastric condition indicated
a
Ibuprofen, ** Paracetamol that no change in analyte chemical form occurred for a period
BORAHAN ET AL.
of 210 min. Spiking experiments were performed to ensure
that there were no shifts in the retention times of analytes.
CONFLICT OF I N T E R E ST
The authors have declared no conflict of interest.
ORC ID
Sezgin Bakırdere https://orcid.org/0000-0001-9746-3682
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How to cite this article: Borahan T, Unutkan T,
Şahin A, Bakırdere S. A rapid and sensitive reversed-
phase HPLC method for simultaneous determination of
ibuprofen and paracetamol in drug samples and their
behaviors in simulated gastric conditions. J Sep Sci
2018;1–6. https://doi.org/10.1002/jssc.201801002