A comparative study for the quantitative

determination of paracetamol in tablets using UV-

Visible spectrophotometry and high performance
liquid chromatography
Murtaza Sayed, M. Ismail, Sanaullah Khan and Hasan M. Khan*
Radiation and Environmental Chemistry Laboratory, National Centre of Excellence in Physical
Chemistry, University of Peshawar-25120, Pakistan
*Corresponding author E-mail: hmkhan3@gmail.com, hmkhan@yahoo.com

Abstract
This study investigates the comparison of high performance liquid chromatography (HPLC) and UV-
Spectrophotometry methods for the quantitative determination of paracetamol in commercially
available tablets. HPLC analysis was carried out on reversed phase XDB-C18 column (4.6 × 150 mm,
5 micron) with UV-Visible detection at 230 nm. The mobile phase used was acetonitrile-water (25:75
% v/v) at a flow rate of 1 mL min-1, column temperature of 40 0C and the determination was
completed in less than 5 min. The method developed for HPLC analysis showed excellent linearity in
the range of 2-20 µg mL-1 with regression coefficient of 0.993 and the lower limit of detection was
found to be 0.829 µg mL-1. The lower limit of detection for UV-Visible Spectrophotometer was found
to be 4 µg mL-1, while linearity was found in the range 4 - 10 µg mL-1. After that range deviations
were observed from Beer’s law. Precision (% R.S.D < 1) and mean recovery was found for UV- visible
spectrophotometer in the range of 99.00 – 100.10 %, while for HPLC recovery was calculated to be in
the range of 99.71 – 100.95 %. The two evaluated methods showed to be adequate to quantify
paracetamol in tablets, while HPLC method presented the most reliable results for the analysis of
tablets. Paracetamol content in tablets was also tested statistically using test hypothesis.

Keywords
Paracetamol, HPLC,UV-Vis Spectrophotometry, Mean recovery, Precision

INTRODUCTION
Paracetamol (p-hydroxy acetanilide) is a compound with analgesic and antipyretic properties and is
commonly used for relief of fever, headaches and other minor aches and pain. The chemical structure
of paracetamol is shown in Fig. 1. Already reported work reveals that many analytical methods have
been proposed for its quantitative determination in paracetamol tablets, like spectrophotometry
(Hoang et al., 2014; Gul et al., 2015), second derivative spectrophotometry (Kokot and Burda, 1997),
planer chromatography (Argekar and Sawant, 199), IR spectroscopy (Bouhsain et al., 1997), capillary
chromatography (Emre and Özaltın, 2007) and high-performance liquid chromatography (Silvana et
al., 2013). This drug is official in Indian pharmacopeia (Riedel and Laufen, 1993), British
pharmacopeia (2006), European pharmacopeia (1997) and United States pharmacopeia (2005). Due
to the rapid growth, scientific development, and industrialization, various synthetic drugs have been
introduced to the market. The increased utilization of these drugs demands the development of fast
and economical analytical methods for their successful analysis in raw materials, pharmaceutical
tablets and in biological samples.
The purpose of the present study was to develop easy and fast analytical methods to quantify
paracetamol in raw materials and tablets. The results obtained by these methods were also compared
statistically.
EXPERIMENTAL
Reagents and Chemicals
Paracetamol (acetaminophen), reference standard was obtained from Stanley Pharmacauticals.
Methanol and acetonitrile (HPLC Grade) were obtained from Merck (Germany). Grade 1 water was
obtained from a Milli-Q ultra pure water purification system. (Millipore, USA). Paracetamol tablets
labelled to contain 40 mg/tablet was purchased from local markets.

Instruments and analytical conditions

The HPLC analysis was carried out using an Agilent 1200 system, composed of quaternary pump, UV
detector and HP Chemstation software. The column used was a symmetry C18 (250 mm x 4.6 mm
i.d., 5 µm particle size) maintained at 40 0C and UV detection was performed at 230 nm. The mobile
phase consisted of acetonitrile and water (25:75 % v/v) at a flow rate of 1 mL min-1 and the injection
volume was 4 µL.
UV-Spectrophotometric analyses were carried out on Perkin Elmer Lambda 650 UV/Vis
spectrophotometer using quartz cuvette having path length of 10 mm. For quantification of
paracetamol, scan mode was selected and measurements were obtained by taking water as blank.
Preparation of standard and reference solutions
Paracetamol standard solution: Approximately 40 mg of paracetamol reference standard was
weighed and transferred to 500 mL flask. One mL of 0.5 N NaOH was added to ensure complete
solubility and the solution was diluted to volume with distilled water. Subsequent dilutions were made
with water to give the concentrations of 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 µg mL-1 of paracetamol.
Paracetamol sample solution: To analyse the paracetamol content in commercially available
tablets, about 10 tablets were accurately weighed, finely powdered and then transferred to 500 mL
flask. 0.5 N NaOH (1 mL) was added for solubility and the solution was then diluted to mark with
distilled water. The solution was filtered with 0.45 µm membrane filter paper prior to analysis by HPLC
and UV-Visible spectrophotometer. Dilutions were then made from stock solution having the
concentrations of 2, 4, 6 and 8 µg mL-1.
Method Validation
Limit of quantification (LOC) and limit of detection (LOD): For measuring LOC and LOD, a series
of diluted standard paracetamol solutions were analysed by both HPLC and UV- visible
spectrophotometer. Limit of detection for paracetamol by HPLC method was calculated at the signal
to noise ratio of 3, and was found to be 0.829 µg mL-1. The limit of quantification of paracetamol was
calculated at signal to noise ratio of 10 and was found to be 2.763 µg mL-1. Limits of detection and
quantification for UV/Vis spectrophotometry were also calculated and were found to be 4.0 µg mL-1
and 13.3 µg mL-1, respectively.
Linearity: Linearity of HPLC method was evaluated by analysis of working standard solutions of ten
different concentrations (n = 3). The regression equation obtained was y = 11.619x - 9.6334 (r2 =
0.993, n = 10). The range of linearity was from 2 to 20 µg mL-1. For UV/Vis Spectrophotometry, the
linearity was calculated in the range from 4 to 18 µg mL-1. In the range of 4 to 10 µg mL-1, the plot
was linear obeying Beer’s law with a coefficient of correlation r2 of 0.9772. Beyond 10 µg mL-1 the
data showed Beer’s law with negative deviation. By applying Q-test at 90 % confidence interval points
beyond 10 µg mL-1 were removed from Fig. 2 and thus a linear plot was obtained as shown in Fig. 3.
Accuracy: The accuracy of the method was determined by adding reference standard of paracetamol
to the tablet samples at three different concentrations of 4, 6 and 8 µg mL-1. Sample solutions were
made in triplicate and were analysed for three consecutive days using HPLC and UV/Vis
spectrophotometer. Solutions for the calibration curve were prepared fresh every day. The assay
accuracy results from the two methods are summarized in Table 2 showing the recovery percentage
of UV and HPLC methods.

Precision: The intra-day precision was calculated by analysing sample solutions of three different
concentrations 4, 6 and 8 µg mL-1 (n = 3) using the UV and HPLC methods. The intermediate
precision (inter-day variation in the same concentration) was calculated for three consecutive days (n
= 3). Paracetamol content and relative standard deviations (R.S.D) for both UV and HPLC methods
were calculated and are summarized in Table 2.
Analysis of Paracetamol tablets
The samples of paracetamol tablets were analysed by HPLC and UV methods. Before the analysis,
the tablets were weighed and finely powdered. The sample solutions for the HPLC and UV analysis
were prepared as described earlier in the Section 2.3.1. The paracetamol contents were determined
using the two methods and the obtained results were statistically proved using test hypothesis at 0.01
significance level.

RESULTS AND DISCUSSION

Different chromatographic conditions were tried by changing the composition of the mobile phase,
flow rate and column. Finally, applying mobile phase of water: acetonitrile in a ratio of (75:25 v/v) at a
flow rate of 1 mL min-1 showed better results. So using this combination of mobile phase at pH 7.4
and a C18 column, an adequate peak symmetry (symmetry = 0.5) and short run time of 3 min was
achieved as shown in Fig.4
In UV-Visible spectrophotometry, the standard paracetamol sample showed an absorption band at
230 nm (Fig 5). So wavelength of 230 nm was selected for absorption measurement of paracetamol
content in the tablets.
Validation
A linear relationship was found between paracetamol concentration and response time of both HPLC
and UV methods. Table 1 shows regression analysis data for both the methods. HPLC shows the
regression coefficient of 0.993 while UV method shows regression coefficient value of 0.9772 that is
little lower than HPLC method.
The precision data obtained for the two methods are tabulated in Table 2. Both HPLC and UV-visible
spectrophotmetric methods showed R.S.D. values lower than 2% presenting good precision, however
HPLC method was more precise than UV method.
Accuracy was investigated by means of % recovery experiments (n = 3) using developed methods.
Both chromatographic and spectrophotometric methods exhibited recoveries close to 100%, however
better recovery was achieved by HPLC-method.
The LOD and LOQ for chromatographic method were obtained by considering signal to noise ratio of
3 and 10 and were found to be 0.829 µg mL-1 and 2.76 µg mL-1, respectively. For the UV-method,
LOD and LOQ were found to be 1.25 µg mL-1 and 3.51 µg mL -1, respectively. The HPLC method
was found to be a more sensitive method as compared to UV method.
Table 1: Overview of the linearity data for paracetamol by the chromatographic and
spectrophotometric methods

HPLC UV
Regression parameters
Regression Coefficient (r2) 0.993 0.977

Slope ± Standard Error 11.619±0.344 0.0455±0.004909

Intercept± standard Error -9.6334±4.27 -0.0024±0.03607

Standard Error of Estimate 6.27 0.021952

Concentration Range (µg mL-1) 2-10_ 4-10_

Number of Points 10 4

Table 2: Validation Parameters of the elevated methods for Paracetamol determination

HPLC UV
Validation Parameter
Intra-day Precision n=3 (R.S.D %) 0.332 0.452

Inter-day Precision n=3 (R.S.D %) 0.281 0.556

Accuracy n=3 (Recovery, %) 99.71-100.95 99.00-100.10

LOD (µg mL-1) 0.829 1.25

LOQ (µg mL-1) 2.76 3.51

Table 2: Quantitative analysis of paracetamol content (Claimed concentration 40 mg/tablet) in
commercially available tablets by HPLC and UV methods

HPLC UV
Factors (n = 5)
Mean (%) 99.95 97.89

Amount found (mg) 39.99 39.99

SD 0.3501 0.4511

R.S.D (%) 0.3503 0.4521

Analysis of Paracetamol content in tablets

The quantitative analysis of paracetamol in tablets was carried out using both HPLC and UV-visble
spectrophotometric methods and is presented in Table 3. Both methods showed the nearly same
amount of paracetamol content in tablet, however HPLC method showed slightly lower % R.S.D than
UV-method. So, both analytical techniques can be employed for quantitative analysis of paracetamol
content in the tablets.

CONCLUSIONS
It can be concluded from the present work that both HPLC and UV methods can be used for the
quantitative analysis of paracetamol in tablets and raw material. However, the chromatographic
method is showed to be more sensitive, accurate and precise than UV method. The main advantage
of the spectrophotometric technique over the chromatographic one is that the UV method is more
simple and does not acquire the use of mobile phase and other procedures normally associated with
the chromatographic techniques. The results showed that paracetamol content in tablets can be
quantitatively determined by using HPLC and UV- spectrophotometric methods and no interference of
recipients in the tablets was observed.
ACHNOWLEDGMENT
The authors are grateful to the Higher Education Commission (HEC) of Pakistan for providing
financial support to carry out this research.

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