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Development and validation of RP-HPLC method for simultaneous estimation

of paracetamol, diclofenac potassium and famotidine in its combined
pharmaceutical dosage form

Article · December 2014

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Research Article

Development and Validation of RP-HPLC Method

for Simultaneous Estimation of Paracetamol,
Diclofenac Potassium and Famotidine in its
Combined Pharmaceutical Dosage Form
ZEENAT A. SAMLAYAWALA, SHAILESH K. KORADIA
Babaria Institute of Pharmacy, Vadodara-Mumbai NH-8,Varnama, Vadodara-391240, Gujarat, India.

ABSTRACT
A simple, precise and accurate isocratic RP-HPLC method have been developed for the simultaneous estimation
of PCM, DCP and FAMO in their combined pharmaceutical dosage form. Chromatographic separation was
achieved on a ODS Hypersil C18 (250 x 4.6 mm i.d., 5μm) column, kept at ambient temperature, using a mobile
phase consisting of 20 mM phosphate buffer (pH 3.5) : methanol (80:20 v/v) at a flow rate of 1.0 mL/min and
UV detection at 217 nm. The linearity was observed in the concentration range of 10-60 μg/mL, 2.5-15 μg/mL
and 1-10 μg/mL for Paracetamol, Diclofenac potassium and Famotidine respectively. The % recovery was
found to be 99.81-101.25%, 99.63-100.85% and 99.63-100.66% for Paracetamol, Diclofenac potassium and
Famotidine respectively. The developed method can be successfully applied in routine quality control analysis.

Keywords: Paracetamol, Diclofenac potassium, Famotidine, RP-HPLC method, simultaneous estimation

INTRODUCTION reduced basal and nocturnal gastric acid secretion. It is

A combination of Paracetamol (PCM), Diclofenac potas-
sium (DCP) and Famotidine (FAMO) is used for the
treatment of joint and muscular pain [1]. PCM [N-(4-
hydroxyphenyl) acetamide] also known as Acetamino-
phen, is commonly used for its analgesic and antipyretic
effects. PCM is act primarily in the CNS, increasing the
pain threshold by inhibiting isoforms of cyclooxygenase,
COX-1, COX-2, and COX-3 enzymes involved in prosta-
glandin (PG) synthesis[2,3]. DCP is a phenylacetic acid
derivative that is a potent inhibitor of COX and that has
analgesic, anti-inflammatory and antipyretic effects.
Chemically it is 2-{2-[(2,6-dichlorophenyl) ami-
no]phenyl} acetic acid and used in the acute and chron-
ic treatment of osteoarthritis and rheumatoid arthri-
tis[3,4]. FAMO is chemically 3-[({2 [(diaminomethyli-
dene) amino]-1,3-thiazol-4-yl} methyl) sulfanyl]-N'-
sulfamoylpropanimi-damide. Blocking histamine affects
by competitively binding to H2-receptors located on the
basolateral membrane of the parietal cell, results in
used for the treatment of peptic ulcer disease (PUD) and
gastroesophageal reflux disease (GERD) and hyperse-
cretoryconditions, such as Zollinger-Ellison syn-
drome[3,5].
Literature review reveals that HPTLC method has been
reported for analysis of PCM, DCP and FAMO in phar-
maceuticals [6]. The methods have been reported in-
cluding UV Spectrophotometry, HPLC, Stability indicat-
ing HPLC, HPTLC and LC-MS for quantitation in formula-
tions[7-19], either alone or in combination with other
drugs. This paper reports simultaneous quantitation of
PCM, DCP and FAM by RP-HPLC in combined pharma-
ceutical dosage form. The proposed method was vali-
dated according to International Conference on Harmo-
nization (ICH) guidelines Q2(R1)[20].
MATERIALS AND METHODS
Reagents
PCM and DCP gifted by Troikaa Pharmaceuticals Ltd,

 Address for correspondence:

Zeenat A. Samlayawala, Babaria Institute of Pharmacy, Vadodara-Mumbai NH-8, Varnama, Vadodara-
391240, Gujarat, India.
E-mail address: zeenat.90@rediffmail.com, Phone: +91 9725956418.
Received: 20/07/2014, Revised: 28/08/2014, Accepted: 10/09/2014

International Journal of Pharmaceutical Research | October-December 2014 | Volume 6 | Issue 4 | 95

 Samlayawala & Koradia / RP-HPLC method for estimation of paracetamol, diclofenac and famotidine
Ahmedabad. FAMO was gifted by Cadila Pharmaceuti- ure 1).
cals, Ahmedabad. Pharmaceutical dosage formulation
was procured from a local pharmacy. HPLC grade
methanol, water, orthophosphoric acid (OPA) and tri-
ethylamine (TEA) (Sd fine chemicals, India); AR grade
Potassium dihydrogen orthophosphate (Sd fine chemi-
cals, India) and nylon membrane filter paper (0.45μ)
were used throughout the practical.
Instrument
HPLC system (model: 1220, Agilent Technologies Ltd,
Japan) having ODS Hypersil C18 (250 mm x 4.6 mm
i.d., 5μm) column, 1220 Infinity binary gradient pump
and DAD detector; attached to a computer software LC-
Lab Chemstation and Double beam UV-Visible spectro-
photometer (Model 1800, Shimadzu Ltd, Japan) with a Figure 1: Overlain spectra of PCM, DCP and
pair of 1 cm matched quartz cells were used. Electronic FAMO (10 μg/mL) in mobile phase.
analytical balance (Model AUX220, Shimadzu, Japan),
Ultrasonic cleaner (Model EIE 808, EIE Instruments Pvt. Optimized chromatographic condition
Ltd., India) and Digital pH meter (Toshcon Industries Pvt. Selection of mobile phase was done based on literature
Ltd., India) were used. All the instruments and glass review, solubility and pKa value of the drugs. The pure
wares were calibrated prior to use. drug solution of PCM, DCP and FAMO were injected into
Chromatographic condition HPLC system and allowed to run in different mobile
phases. Various mobile phases were tried in order to
The chromatographic separation was achieved on ODS find optimum condition for the separation of three com-
Hypersil C18 (250 mm x 4.6 mm i.d., 5μm) column at pounds. (Figure 2)
ambient temperature. The mobile phase was comprised
of 20 mM phosphate buffer (pH 3.5):metahnol (80:20)
was pumped at a flow rate of 1.0 mL/min and effluents
were monitored at 217 nm. The mobile phase was fil-
tered through 0.45μm membrane filter and degassed
before used.
Preparation of mobile phase
Accurately weighed 2.72 gm of potassium dihydrogen
orthophosphate and 2 mL of TEA dissolved in 800 mL of
water, adjusted pH 3.5 with OPA and diluted to 1000
mL of water. Figure 2: Optimized Chromatogram of standard
DCP, PCM and FAMO at 217 nm.
Preparation of standard stock solution
Accurately weighed PCM (25 mg), DCP (25 mg) and Method validation
FAMO (25 mg) were transferred in to 25 mL volumetric
The developed method was validated as per ICH guide-
flask separately, dissolved and diluted up to the mark
line (Q2-R1) in terms of validation parameters like line-
with methanol to obtained standard stock solution of
arity and range, precision, accuracy, Limit of Detection,
1000μg/mL of PCM, DCP and FAMO. An aliquot of 2.5 Limit of Quantitation, robustness and specificity.
mL of stock solution of each was transferred into 25 mL
volumetric flask, separately and diluted to mark with Linearity
mobile phase to obtain standard solution of 100 μg/mL. Linearity of the method was determined by conducting
These solutions were used as working standard solu- calibration curve at five independent levels in the rang-
tion. The solutions were protected from light. es of 10-60 μg/mL, 2.5-15 μg/mL and 1-10 μg/mL
Selection of detection wavelength PCM, DCP and FAMO respectively. The calibration
curves were constructed by plotting peak area (mAU*s)
Standard solutions (10 μg/mL) of PCM, DCP and FAMO versus concentration (µg/mL) of PCM, DCP and FAMO
were scanned individually over 400-200 nm range and regression equation were calculated.
against mobile phase as a blank. It was observed that
three drugs showed considerable absorbance at 217 LOD and LOQ
nm was selected as the wavelength for detection (Fig- LOD and LOQ were calculated from calibration curve by

96 | International Journal of Pharmaceutical Research | October-December 2014 | Volume 6 | Issue 4

 Samlayawala & Koradia / RP-HPLC method for estimation of paracetamol, diclofenac and famotidine
using the following equations as
per ICH guidelines.
LOD = 3.3 x (σ/S) and LOQ = 10 x (σ/S)
Where, σ = the standard deviation of Y- intercept of
regression line and S = the slope of calibration curve
Method Precision (Repeatability)
Repeatability study was performed by analyzing mixed
solution of PCM, DCP and FAMO (50, 5 and 2 μg/mL) for
six times by developed RP-HPLC method and %RSD
was calculated.
Intermediate precision (Reproducibility)
The intraday and interday precision studies were carried
out by analysis of three solutions three times on the
same day and three different days. Three different solu-
tions containing PCM (10, 30 and 50 μg/mL), DCP (2.5,
7.5 and 12.5 μg/mL) and FAMO (1, 4 and 8 μg/mL) and
% RSD was calculated.
Accuracy (% Recovery)
The proposed method was applied to determine recov-
ery of PCM, DCP and FAMO from tablet formulation. The
recovery study was carried out in triplicate by spiking
standard PCM, DCP and FAMO in to prequantified sam-
ple solution of 25, 2.5 and 1.0 µg/mL (PCM, DCP and
FAMO) at 80%, 100 % and 120 %. The chromatograms
were recorded & from the peak area of drug, % recov-
ery was calculated from regression equation of the cali-
bration curve.
Robustness
Robustness study was performed in following altered
chromatographic conditions:
Variation in mobile phase
Variations in the flow rate (± 0.1 mL/min)
Variation in pH of mobile phase (± 0.2)
Three time injections of a 20 µL solution of PCM, DCP
and FAMO (50, 5, 2µg/mL) were analyzed as per chro-
matographic conditions in each altered condition and
chromatograms were recorded.
Specificity
The method was determined as specific by comparing
test results obtained from analyses of sample solution
containing placebo ingredients with that of test results
those obtained from analysis of standard solution.
a) Blank mobile phase
b) Standard mixture of PCM, DCP and FAMO (50, 5, 2
µg/mL)
International Journal of Pharmaceutical Research | October-December 2014 | Volume 6 | Issue 4 | 97
c) Sample mixture of PCM, DCP and FAMO (50, 5, 2
µg/mL)
Analysis of marketed formulation
Twenty tablets were weighed and finely powdered. Tab-
let powder equivalent to 500 mg of PCM, 50 mg DCP
and 20 mg FAMO was transferred in to a 100 mL volu-
metric flask. Then 80 mL of methanol was added and
sonicated for 10 min., volume was made up to mark
with methanol. This solution is expected to contain
5000, 500 and 200 μg/mL of PCM, DCP and FAMO re-
spectively. The solution was filtered through Whatman
filter paper. Aliquot of 5 mL of resultant solution was
transferred into a 50 mL volumetric flask and diluted up
to the mark with mobile phase to get concentration of
500, 50 and 20 μg/mL of PCM, DCP and FAMO respec-
tively. Aliquot of 5 mL of resultant solution was taken
into a 50 mL volumetric flask and diluted up to the mark
with mobile phase to get a final concentration of 50, 5
and 2 μg/mL of PCM, DCP and FAMO respectively. The
sample solution of 20 µL was injected under optimized
chromatographic condition and % content of PCM, DCP
and FAMO in the sample mixture was calculated using
regression equation (Table 2)
RESULTS AND DISCUSSION
Method validation
Linearity
The linearity for PCM, DCP and FAMO were found to be
in the range of 10-60 μg/mL, 2.5-15 μg/mL and 1-10
μg/mL respectively.
LOD and LOQ
The LOD and LOQ were found to be 0.043 and 0.130
μg/mL for PCM, 0.074 and 0.224 μg/mL for DCP and
0.034 and 0.103 μg/mL for FAMO respectively.
Method Precision
Repeatability
%RSD was found to be 0.694, 0.387 and 0.859 for
PCM, DCP and FAMO respectively.
Intermediate precision (Reproducibility)
Intraday precision: %RSD was found to be 0.524,
0.433 and 0.410 for PCM, DCP and FAMO respective-
ly.
Interday precision: %RSD was found to be 0.650,
0.706 and 0.805 for PCM, DCP and FAMO respectively.
Accuracy (% Recovery)
Accuracy of the method was confirmed by recovery
study in marketed formulation at three level of standard
addition. Percentage recovery for PCM, DCP and FAMO
was found to be within accepted limit (98-102%).
 Samlayawala & Koradia / RP-HPLC method for estimation of paracetamol, diclofenac and famotidine
Table 1: Summary of validation parameters.
Results
Parameters
PCM DCP FAMO
Linearity (μg/mL) 10-60 2.5-15 1-10

Regression equation
y = 31.131x + 26.213 y = 6.121x + 34.730 y = 47.778x + 6.515
(y = mx + c)
31.131 6.121 47.778
Slope(m)
26.213 34.730 6.515
Intercept (c)
Correlation coefficient (r2) 0.9990 0.9994 0.9996
LOD (μg/mL) 0.043 0.074 0.034
LOQ (μg/mL) 0.130 0.224 0.103
Precision (%RSD)
a) Repeatability (n=6) 0.694 0.387 0.859
b) Intraday (n=3) 0.051 0.579 0.338
c) Interday (n=3) 0.140 0.742 0.629
Accuracy (%Recovery)
80% 98.87 ± 0.589 99.63 ± 1.328 100.04 ± 0.641
100% 101.26 ± 0.702 100.81 ± 0.427 100.66 ± 0.750
120% 99.81 ± 0.675 100.85 ± 0.725 99.63 ± 1.062
Robustness Robust
Specificity Specific

Robustness
Robustness was checked by changing the flow rate, pH
of mobile phase and mobile phase composition. The
%RSD for robustness determination was found to be
less than 2%.
Specificity
There was no interference of mobile phase and excipi-
ents at the retention time of PCM, DCP and FAMO (Fig-
ure 3-5).
Figure 4: Chromatogram of standard DCP,
PCM and FAMO at 217 nm.

Figure 3: Chromatogram of blank mobile phase

at 217 nm.
Figure 5: Chromatogram of sample DCP, PCM
Analysis of marketed formulation and FAMO at 217 nm.
The applicability of the method was tested by
CONCLUSION
analyzing the commercially marketed tablet
formulation. The results were shown in table 2. The developed RP-HPLC technique is simple, precise

98 | International Journal of Pharmaceutical Research | October-December 2014 | Volume 6 | Issue 4

 Samlayawala & Koradia / RP-HPLC method for estimation of paracetamol, diclofenac and famotidine
and accurate. The method is suitable for the analysis of Application to Pharmaceuticals, 2013, Analytical
PCM, DCP and FAMO as bulk drugs and in a pharma- Chemistry Insights, 8 : 73–81.
ceutical formulation without any interference from the 11. Mehta K, kumar B and Dubey A, Development and
excipients. validation for simultaneous Estimation of famotidine
and Diclofenac Poatassium in combined Tablets
Table 2: Analysis of marketed formulation dosage form by First order derivative method, 2012,
% Drug found
Label claim Amount found International Journal of Research in Pharmacy and
Drug Mean± SD
(mg) (mg)
(n=6)
Chemistry, 2(4) : 1023-1028.
12. Gowramma B, Rajan S, Muralidharan S,
PCM 500 509.170 101.834 ± 0.719
Meyyanathan S and Suresh B, A validated RP-
DCP 50 50.075 100.152 ± 0.828 HPLC method for Simultaneous estimation of
FAMO 20 19.862 99.309 ± 0.911 paracetamol and Diclofenac potassium in
pharmaceutical Formulation, 2010, International
Journal of ChemTech Research, 2(1) : 676-680.
ACKNOWLEDGMENTS 13. Sindhur Nag N, Gouthami B, Madhuri L, Krishnaveni
We would like to thank Troikaa Pharmaceuticals Ltd and N, Meyyanathan S N and Suresh B, Development
Cadila pharmaceuticals, Ahmedabad, for providing gift and validation of a RP-HPLC method for the
sample of PCM, DCP and FAMO. simultaneous determination of paracetamol and
diclofenac potassium on stainless steel surface of
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International Journal of Pharmaceutical Research | October-December 2014 | Volume 6 | Issue 4 | 99

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