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[Research article]
A validated RP-HPLC method for the simultaneous estimation of
paracetamol and naproxen in bulk and tablet dosage forms
*Krishanu Pal1, V.Murali Balaram2, M.Bhagavan Raju3
*1Dept. of Pharmaceutical Analysis, SriKrupa Institute of Pharmaceutical Sciences, Siddipet-
502276, AP.
2
Dept. of Pharmaceutical Analysis, Sultan-Ul-Ulum College of pharmacy, Hyderabad, AP.
3
Dept. of Pharmaceutical Chemistry, Gland Institute of Pharmaceutical Sciences, Hyderabad,
AP.
.
ABSTRACT
A simple, sensitive, accurate, precise, reproducible, specific and cost-effective RP-HPLC method was developed
and validated for the simultaneous estimation of Paracetamol (PCM) and Naproxen sodium (NPX) in the bulk
and pharmaceutical dosage forms. Chromatography was carried on a Nucleosil C 8 (250 X 4.6 mm, 5μm) column
with mobile phase comprising of phosphate buffer pH-6.5 adjusted with 1N potassium hydroxide and
acetonitrile in the ratio of 70:30 v/v. The flow rate was adjusted to 1.0 ml/min with UV detection was carried
out at 240 nm, and column temperature of 30±0.50 C was maintained throughout the study. The retention times
for PCM and NPX were found to be 3.54 min and 4.59 min respectively. Linearity values were obtained in the
range of 2.5-15 µg/ml for PCM and 2-12 µg/ml for NPX with correlation coefficient (r2) of 0.999 for both drugs.
The method was validated as per ICH guidelines for specificity, linearity, precision, accuracy, robustness, limit
of detection and limit of quantification. So the proposed method was found to be suitable for the routine quality
control analysis of these drugs in raw materials and also in pharmaceutical dosage forms.
Keywords: RP-HPLC, Paracetamol or PCM and Naproxen sodium or NPX, Nucleosil C 8 column, phosphate
buffer pH 6.5, and acetonitrile.
INTRODUCTION
Non-steroidal anti-inflammatory drugs (NSAIDs) from arachidonic acid by the enzyme
are among the most frequently prescribed drugs cyclooxygenase which exists in a constitutive
worldwide and are used for mainly analgesic, (COX-1) and an inducible (COX-2) isoforms. Most
antipyretic and anti-inflammatory actions. The of the NSAIDs inhibit COX-1 and COX-2 non-
main mechanism of action behind the anti- selectively, but some selectively inhibits COX-2.
inflammatory activity of major NSAIDs is PCM is a para-amino phenol derivative comes
blockage of prostaglandins (PGs), as generation of under the classification of broad group of
PGs locally at the site of inflammation mediate nonsteroidal anti-inflammatory drugs which is
many of the inflammatory changes. Prostaglandins, chemically N-(4-Hydroxyphenyl) acetamide
prostacyclin and thromboxane-A2 are produced (Figure 1a). It is one of the most popular drugs
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* Corresponding author: Krishanu Pal
E-mail address: krishanupal5227@gmail.com
Krishanu Pal et al / Int. J. of Pharmacy and Analytical Research Vol-2(4) 2013 [184-193]
under this category and has good analgesic and It works by reducing PGs synthesis by inhibiting
antipyretic properties but mild anti-inflammatory the enzyme cyclooxygenase, both COX-1 and
activity. It is commonly used for the relief COX-2 enzymes that cause inflammation and pain
of headaches and other minor aches and pains, and in the body. It is clinically proven as the most
is a major ingredient in numerous cardio safe NSAID with reduced side effects like
cold and flu remedies. It works by blocking the considerable hepatic and renal safety. This
action of the enzyme cyclooxygenase which is combination of PCM and NPX exhibits anti-
responsible for the production of PGs. NPX is a inflammatory, analgesic and antipyretic activities.
propionic acid derivative related to the arylacetic PCM has central action and NPX has peripheral
acid group of nonsteroidal anti-inflammatory drugs action. Thus the combination is more effective
which is chemically (2S)-2-(6-methoxynaphthalen- when individual drugs act by different mechanism
2-yl) propionic acid (Figure 1b). It is commonly and act synergistically. By activating multiple pain
used to treat pain or inflammation caused by acute inhibitory pathways PCM and NPX combination
masculo-skeletal pain, headaches and migraines, has more effective pain relief property and at the
and conditions such as arthritis, ankylosing same time decreased adverse effects.
spondylitis, tendinitis, bursitis, gout, or menstrual
cramps.
Literature survey shows that there are several UV control, and a PDA detector using Waters
spectrophotometric, HPLC, LC-MS and HPTLC (Alliance) Empower-2 software. For normal UV
methods been reported for estimation of PCM and absorbance estimations during trials were done by a
NPX individually in bulk drugs and also in Lab India UV- VIS spectrophotometer UV 3000+,
respective formulations. Even there are many UV, and weighing of drugs and chemicals were done by
HPLC and HPTLC methods reported for estimation a Shimadzu precision balance AUX-220.
of PCM and NPX in combination with other drugs
in various formulations. But there are three UV Chemicals and reagents
spectrophotometric, one HPLC and one UPLC Paracetamol and Naproxen sodium RSwere
method been reported till now for simultaneous obtained as a gift samples from Aurbindo Pharma
estimation of PCM and NPX in fixed dose Ltd, Hyderabad. Due to unavailability of the Mkd.
combination formulations. So in the present work formulation in the local market during the time of
an attempt was made for developing a simple, experimentation PCM and NPX synthetic mixture
sensitive, accurate, precise, reproducible, specific was prepared by placebo technique and formulated
and cost-effective RP-HPLC method for the as tablet. HPLC grade acetonitrile and water were
simultaneous estimation of PCM and NPX in the procured from Merck (Mumbai, India) and
bulk drug and pharmaceutical dosage form, and potassium dihydrogen phosphate , dipotasssium
also to validate the same as per ICH guidelines. hydrogen phosphate and potassium hydroxide of
analytical grade were used for the studies. The
EXPERIMENTAL solvents and mobile phases after preparation were
Instrumentation filtered using Millipore 0.45 µm filter medium.
A Waters Alliance 2695 Separation Module-HPLC
system comprising of quaternary, low pressure Chromatographic conditions
mixing pump and inline vacuum degasser with
A Nucleosil C8 (250 X 4.6 mm, 5μm) column was
auto sampler and programmable temperature
used as a stationary phase, and phosphate buffer
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Krishanu Pal et al / Int. J. of Pharmacy and Analytical Research Vol-2(4) 2013 [184-193]
comparing chromatograms of blank, individual and After observing a steady base line with the
mixed reference standards and formulation tablets. optimized chromatographic conditions, three mixed
A blank solution (mobile phase) was injected and drug dilutions of PCM and NPX bulk drugs each
the chromatogram showed no interfering peaks at having 10 µg/ml and 8 µg/ml of PCM and NPX
the retention time of both the drugs. The respectively were prepared and injected and the
chromatograms of PCM and NPX extracted from chromatograms were recorded. The procedure was
the tablets were compared with the chromatograms repeated for the tablet sample dilutions having 10
acquired from PCM and NPX reference standards; µg/ml and 8 µg/ml of PCM and NPX respectively.
correlation was found good in terms of Rt and peak Finally using the peak area values of PCM and
area which indicates specificity of method. NPX standard drug dilutions the % purity of the
bulk drug and tablet formulation was calculated.
Linearity and Range
Aliquots of standard stock solutions of PCM and Accuracy
NPX were taken in 10 ml volumetric flasks and To ensure the reliability (accuracy) of the method,
diluted with diluent to get final concentrations in recovery studies were carried out by mixing
range of 2.5-15 μg/ml for PCM and 2-12 μg/ml for standard quantity of standard drug with the pre-
NPX. Triplicate injections were made five times analyzed sample formulation and the contents were
for each concentration for each drug separately. reanalyzed by the proposed method. The accuracy
The peak areas of the chromatograms were plotted of the method was determined by calculating
against the concentrations for PCM and NPX both recoveries of PCM and NPX by standard addition
to obtain the respective calibration curves. method. In this known amount of standard
solutions of PCM and NPX (50%, 100% and 150%
LOD and LOQ spike level) added to previously spiked tablet
The limit of detection (LOD) and limit of sample dilutions containing PCM and NPX were
quantification (LOQ) were estimated from the set prepared in triplicate. A mixed standard dilution of
of 5 calibration curves obtained from serial 5: 4 µg/ml of PCM and NPX was prepared, and
dilutions of PCM and NPX working standard 50%, 100% and 150% from this solution was
solution in order to obtain signal-to -noise ratio of individually added to diluted sample formulation
3:1 for LOD and 10:1 for LOQ. The LOD and having 5: 4 µg/ml of PCM and NPX respectively,
LOQ may be calculated as – and analyzed.
LOD = 3.3XSD/Slope and
LOQ = 10XSD/Slope Ruggedness
Where, The ruggedness of an analytical method is the
SD= standard deviation of the Y-intercepts of the 5 degree of reproducibility of the test results
calibration curves. Obtained by the analysts of the same samples under
Slope= Average of slopes of the 5 calibration a variety of normal test conditions such as different
curves. laboratories, different analysts using same
operational and environmental conditions that may
Precision differ but are still within the specified parameters
The precision study was carried out as both system
of the assay. Ruggedness of the proposed method
and method precisions. The repeatability was
was determined by analysis of aliquots of the
carried out by performing assay of six dilutions of
sample solution having 10 µg/ml and 8 µg/ml of
test sample preparation each having 10 µg/ml and 8
PCM and NPX respectively by two analysts using
µg/ml of PCM and NPX respectively and from this
same operational conditions.
% RSD was calculated (intraday). Intermediate
precision of the method was checked by
Robustness
performing same procedure on a different day
Robustness of the method was determined by
(interday) by another person under same
small, deliberate changes in flow rate, mobile phase
experimental conditions.
ratio and detection wavelength. Typical changes
include flow rate changing to 1.0±0.1 ml/min,
Assay study column temperature change ±20C and detection
wavelength change to 240±1 nm.
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Result and discussion 4.595±0.004 for NPX with flow rate of 1.0 ml/min
The present study was carried out with a view to and detection wavelength at 240 nm. With the
develop a simple, accurate, precise and optimized chromatographic conditions method
economical RP-HPLC method for simultaneous validation parameters were performed for
estimation of PCM and NPX in bulk drugs and specificity, linearity, accuracy, precision, limit of
pharmaceutical dosage forms. In order to achieve detection, limit of quantification, robustness and
optimum separation of the component peaks, ruggedness, and analyzed.
mixture of acetonitrile with phosphate and acetate
buffer with different combinations were tested on Specificity
a C-18 type stationary phase. Finally a mixture of Specificity of the method was assessed by
phosphate buffer pH-6.5 adjusted with 1N comparing chromatograms of blank, individual and
potassium hydroxide and acetonitrile in the ratio of mixed reference standards and formulation tablets,
70:30 v/v was selected as the most suitable mobile having a concentration of 10 µg/ml of PCM and 8
phase, as the chromatographic peaks were well µg/ml of NPX. Respective chromatograms were
defined and resolved with no tailing. The retention compared for retention time, resolution factor and
times were obtained 3.550±0.003 for PCM and purity.
Figure-2: A typical chromatogram showing peaks of PCM and NPX in mixed standard solution
Table no-1: Specificity study for PCM and NPX in reference and tablet drug.
4. Mixed 10 8 3.552 4.593 268932 240352 4.09 4008 4558 1.14 1.10
Standard
5. Tablet 10 8 3.551 4.590 269132 239894 4.11 4013 4569 1.13 1.10
Drug
% RSD 0.0162 0.0548 0.9356 0.1418 0.3449 0.8404 0.5279 0.5095 0
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Table no-2: Linearity values of PCM and NPX in mixed standard dilutions.
Serial No. Concentration of Drug in µg/ml Retention time (Rt) Peak Area
PCM NPX PCM NPX PCM NPX
1. 2.5 2 3.545 4.606 65971 58220
2. 5.0 4 3.541 4.587 134197 119645
3. 7.5 6 3.548 4.590 203598 180001
4. 10 8 3.553 4.599 269967 240927
5. 12.5 10 3.559 4.604 337045 300212
6. 15 12 3.590 4.676 415282 369206
Table no-4: Repeatability study of PCM and NPX in mixed standard dilutions.
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Assay
The assay study for PCM and NPX was performed for bulk and tablet formulations, and % purity values were
calculated, which were found to be within the acceptance limit of 97-103%.
Figure-3: A typical chromatogram showing peaks of PCM and NPX in tablet sample dilution
Table no-5: Assay results of PCM and NPX in bulk and tablet formulation dilutions.
Table no-5: Recovery study of PCM and NPX by standard addition method.
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Table no-6: System suitability results for PCM and NPX mixed standard dilutions.
Ruggedness
Ruggedness of the proposed method was NPX respectively by two analysts using same
determined by analysis of aliquots of the sample operational conditions, and the data obtained was
solution having 10 µg/ml and 8 µg/ml of PCM and given in the table-6.
Table no-6: Ruggedness study for PCM and NPX in sample dilutions.
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Table no-7: Robustness study for PCM and NPX in sample dilutions.
Operational Retention Peak Area USP USP Tailing USP Plate count
conditions time Resolution (N)
(Rt) (Rs)
PCM NPX PCM NPX PCM NPX PCM NPX PCM NPX
F1 3.241 4.197 246529 219708 4.16 1.22 1.22 3947 5021
Change in
Flow rate
ml/min)
(±0.2
(±20C)
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