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International Journal of Chromatographic Science

Universal Research Publications. All rights reserved

Original Article
QUANTITATIVE DETERMINATION OF PARACETAMOL AND CAFFEINE
FROM FORMULATED TABLETS BY REVERSED PHASE-HPLC
SEPARATION TECHNIQUE
R. Chandra1, K. Dutt Sharma
Department of Pharmaceutical Chemistry, Dolphin P.G. Institute of Biomedical and Natural Sciences, Dehradun-248001,
Uttarakhand, INDIA
*Corresponding author. Email: rajvimalchandra@gmail.com
Received 10 July 2013; accepted 20 August 2013
Abstract
This RP-HPLC method has been described for the determination binary formulated tablets of paracetamol and caffeine.
However, none have been cross validated using formulated products. The present study depicts an accurate, stable, rapid,
simple, precise and reducible reversed-phase high performance liquid chromatographic (RP-HPLC) separation method.
The present isocratic method was carried out on C18-column with mobile phase methanol and water in the ratio of 40:60 (by
volume) at the flow rate 1.0 mL/minute. The detection was carried out at λ max=243 nm. The retention time of paracetamol
and caffeine were 3.03 and 4.23 minutes, respectively. Under the optimal condition, the linearity were found for
paracetamol R2=0.999 and for caffeine R2=0.994. The limit of detection and limit of quantification for paracetamol were
computed 0.04 and 0.12 µg/mL and for caffeine 0.05 and 0.15 µg/mL, respectively. The recoveries of paracetamol and
caffeine were in the range of 99.62-99.45 % and 104.48-100.56 %. The proposed method was successfully validated to
determine paracetamol and caffeine from its formulated tablets.
© 2013 Universal Research Publications. All rights reserved
Key words: Development, Paracetamol, Caffeine, RP-HPLC, Validation
1. INTRODUCTION 99.7%pure) was purchased from Ranbaxy Pharmaceutical
Paracetamol is used as antipyretic, analgesic and anti- Ltd. Tablets of paracetamol and caffeine with 250 mg and
inflammatory. Chemically, it is 4-Hydroxy Acetanilide. 100 mg purchased from Lupin Pharmaceutical Ltd. HPLC
This is available in combination form in different grade methanol and water were purchased from Merck
formulations. The antipyretic, analgesic and anti- India Limited and 0.45µm nylon membrane filter was
inflammatory effect of paracetamol is due to inhibiting supplied by Pall life Sciences, Mumbai.
prostaglandin synthesis cyclooxygenase-1(COX-1) and 2.2 Instrumentation
cyclooxygenase-2 (COX-2) [1-3]. It is prevent to fever, The instrument, a reversed phase-high performance liquid
headaches, pain of arthritis, aches, colds, flu and period chromatograph was used for method development. The
pain. Paracetamol in combination form with caffeine used chromatograph consisting, a binary pump, column
in migraine attack, child birth and avoid postpartum thermostat and a UV-detector. The eluent was analyzed
hemorrhage [4]. Caffeine is the most wanted drug of the using a C18 column at wavelength (λmax) 243 nm with
world [5]. The chemical name of caffeine is 1, 3, 7- mobile phase methanol and water in the ratio of 40:60 (by
trimethylxanthine. Caffeine is used as a stimulant, due to volume). Injection volume was used 20 µL. The mobile
acts on the central nervous system [6]. Paracetamol in phase was filtered through 0.45µm nylon filter membrane
formulated combination form with caffeine and other drugs followed by sonicate for five min prior to use.
has been validated various methods with spectrophotometry 2.3 Preparation of Stock Solution
and high performance liquid chromatography [7-14]. The Stock solution containing 250 µg/mL of paracetamol and
main aim of this study was to develop a new RP-HPLC 100 µg/mL of caffeine were prepared separately in mobile
method for determination of paracetamol and caffeine from phase. The stock solutions were filtered through a 0.45 µm
formulated tablets, in accordance with International nylon membrane followed by sonicate for five minutes.
conference harmonized guidelines [15]. Serial dilutions were prepared by appropriate dilution of the
2. EXPERIMENTAL stock solutions with mobile phase.
2.1 Chemicals and Reagents 2.4 Methods Validation
Paracetamol and caffeine reference standard (label claim The methods were validated according to ICH guidelines
International Journal of Chromatographic Science 2013, 3(2): 31-34
31
with respect to linearity, specificity, precision, and system
suitability test, limit of detection (LOD) and limit of
quantification (LOQ) [15].
2.5 Linearity
For the measuring linearity were prepared serial dilutions
of 5, 10, 20, 25, 50 and 100 µg/mL from the stock solution
of paracetamol and caffeine. A total volume of 10 mL was
maintained with mobile phase methanol and water. These
different serial dilutions were filtered through a 0.45µm
nylon membrane and sonicate. The each solution of 20 µL
was injected into the column in thrice. The calibration
curves were obtained by plotting peak areas versus known
concentrations in µg/mL.
2.6 Specificity
Specificity was determined with exepient of formulated
tablets. An equivalent weight was taken and solution
prepared similarly to the sample solution. The prepared
solution was determined as per the described method. After
determination was not reported any interference with
exepient at the retention time of the peaks of paracetamol
and caffeine. Therefore, it is concluded that the method is
specific.
2.7 Accuracy
The accuracy of the method was determined by recovery
method. The recovery was checked at the five theoretical
concentrations levels 10, 20, 40, 50 and 100 µg. The
chromatograms were recorded and the percentage recovery
was calculated.
2.8 System Suitability test
The Reproducibility of sample was checked of the system
to measurement of peak area and was carried out using
three replicates of same concentration of standard and
sample, respectively.
2.9 Limit of Detection (LOD) and limit of
Quantification (LOQ)
It is a lowest response of the instrument at lowest
concentration, so the detectable signal value called as limit
of detection and quantifiable noise value called as limit of
quantification. It was measured by signal to noise ratio. To
determine the limit of detection (LOD) and limit of
quantification (LOQ), to prepare three replications [16-19]
of low concentrations serial dilutions of mixed standard of
paracetamol and caffeine from the standard stock solution.
3. Results and Discussion
3.1 Selection of mobile phase
It was a basic need of liquid chromatography. To select the
mobile phase various composition of mobile phases were
checked with water and methanol in different ratio 25:75,
35:65 and 45:55 (by volume) on C18 column at wave length
243 nm and methanol and water 20:80, 30:70 and 40:60 (by
volume) on C18 column at wave length 243 nm. The mobile
phase methanol and water in the ratio of 40:60 (by volume)
was selected. At this mobile phase was obtained suitable
retention time and peak area of both drugs with better
resolution.
3.2 Chromatographic Conditions
Under optimal experimental conditions, a mobile phase
methanol and water in the ratio of 40:60 (by volume), and a
flow rate of 1.0 mL/min, paracetamol and caffeine depicted
a well defined chromatographic separation (resolution
International Journal of Chromatographic Science 2013, 3(2): 31-34
32
factor more than 1.423±0.07) within a run time of 6 min.
The retention times of paracetamol and caffeine 3.03
minutes±0.36 and 4.23 minutes±0.39, respectively. Figure
1 depicts chromatograms of paracetamol and caffeine in
binary mixture.
Figure 1. Chromatogram of binary mixture
3.3 System suitability test
To establish the chromatographic conditions were
performed system suitability test (SST) during the
development and optimization of the method. The test was
performed by injecting the standard mixture in triplicate
and the various parameters retention time, tailing factor,
resolution factor and theoretical plates were computed as
reported by USP [20] and International conference
harmonized guidelines. System suitability parameters were
shown in table 1.
3.4 Linearity
Linearity was determined by the regression analysis. The
calibration curves were plotted between known
concentrations and average peak areas. The method was
linear with a correlation co-efficient (R2) 0.999 and caffeine
having correlation co-efficient (R2) 0.994. The result was
shown in table 2.
3.5 Limits of Detection (LOD) and Limit of
Quantification (LOQ)
The limit of detection (LOD) and limit of quantification
(LOQ) were calculated using the signal-to-noise-ratio of 3
and 10. The limit of detection (LOD) and limit of
quantification (LOQ) values were found for paracetamol
0.04 and 0.12 µg/mL and for caffeine 0.05 and 0.15 µg/mL,
respectively.
3.6 Specificity
The specificity of the method was determined by checking
the interference with the components from placebo. No
interference was observed for any of the components like
excipients of both drugs (figure 1).
3.7 Accuracy
The accuracy of the method was computed by
determination of recovery for five concentrations. The
amount of paracetamol and caffeine recovered and then
percentage of drug content calculated. The mean recovery
Tab. 1. Statistical Summary of validation system suitability parameters.
Paracetamol Caffiene
Parameters
Mean SE Mean SE
Retention time 3.03 minutes ± 0.36 4.23 minutes ± 0.39
Tailing factor 0.82 ± 0.06 0.81 ± 0.07
Resolution factor 1.42 ± 0.07 1.33 ± 0.06
Theoretical plates 475.34 ± 0.44 2012 ± 0.05
SE= Standard Error (±)
Tab. 2. Statistical summary of Linearity, limit of detection and limit of quantification.
Parameters Paracetamol Caffeine
Number of samples per curve 6 6
Correlation range (µg/mL) 10-100 10-100
Regression equation Y=4629x+34584 Y=860.4x-1056
Regression coefficient R2=0.999 R2=0.994
Limit of detection (µg/mL) 0.04 0.05
Limit of quantification(µg/mL) 0.12 0.15
Tab. 3. Recovery experiment for paracetamol and caffeine
Paracetamol Caffeine
Added in µg Found Recovery % Added in µg Found Recovery %
10 9.96 99.62 10 10.44 104.48
20 19.14 95.72 20 20.93 104.68
40 39.9 99.96 40 38.21 95.53
50 51.4 102.89 50 49.83 99.67
100 99.45 99.45 100 100.56 100.56
Mean=99.53 Mean=100.98
SD=2.55 SD=3.79
CV%=2.56 CV%=3.75
SD=standard deviation
CV%=coefficient variation percentage
of paracetamol and caffeine were found 99.53 % and 3. The Indian pharmacopoeia. Published by the Indian
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Source of support: Nil;
Conflict of interest: The authors declare no conflict of interest.
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