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HPLC METHOD FOR DETERMINATION OF PARACETAMOL IN

PHARMACEUTICAL FORMULATIONS AND ENVIRONMENTAL WATER
SAMPLES

Article · January 2018

DOI: 10.20959/wjpr201815-12814

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 World Journal of Pharmaceutical Research
Omar et al. SJIF Impact
World Journal of Pharmaceutical Factor 8.074
Research
Volume 7, Issue 15, XXX-XXX. Research Article ISSN 2277– 7105

HPLC METHOD FOR DETERMINATION OF PARACETAMOL IN

PHARMACEUTICAL FORMULATIONS AND ENVIRONMENTAL
WATER SAMPLES

Nief Rahman Ahmad1 and Farha Khalaf Omar*2

1
Environmental College, Department of Technology, University of Mosul-Iraq.
2
Department of Chemistry, Education College for Girls, University of Mosul-Iraq.

Article Received on
ABSTRACT
13 June 2018, A simple, precise, rapid, and accurate reversed – phase high
Revised on 03 July 2018,
Accepted on 23 July 2018 performance liquid chromatography method has been developed for
DOI: 10.20959/wjpr201815-12814 the determination of paracetamol in pure from, pharmaceutical
formulations and environmental water samples. Chromatography was

*Corresponding Author carried out on supelco L1 (C18) reversed- phase column (25cm ×
Farha Khalaf Omar 4.6mm), 5 microns, using a mixture of acetonitril : buffer pH3.0 (40:
Department of Chemistry, 60v/v) as a mobile phase at a flow rate of 1.5 ml.min-1. Detection was
Education College for Girls,
performed at 243nm at ambient temperature. The retention time was
University of Mosul-Iraq.
found 2.2 minutes. The calibration curve was linear (r= 0.999) over a
farhakhalaf8@gmail.com
niefpharma@yahoo.com concentration range from 10 to 100 µg/ml. Limit of detection (LOD)
and limit of quantitation (LOQ) were found 3ng/ml and 9 ng/ml
respectively. The method was validated for its linearity, precision and accuracy. The
proposed method was successfully applied for the determination of paracetamol in pure form,
pharmaceutical formulations and in environmental water samples.

KEYWORD: HPLC, Paracetamol, Pharmaceutical Formulations, Environmental Water

Samples.

INTRODUCTION
Paracetamol (acetaminophen or N-acetyl-4-aminophenol), is a popular analgesic and
antipyretic agent, with the following structural formula, Figure (1).[1,2]

Molecular formula: C8H9NO2ı= 151.2

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Omar et al. World Journal of Pharmaceutical Research

Figure [1]: Chemical structure of Paracetamol.

Several analytical methods have been devised for the determination of paracetamol. These
methods include titrimetric method[3], HPLC methods[4,6], HPTLC methods[7,8], LC
methods[9], Uv- visible spectrophotometric methods[10,13], amperometric method[14],
fluorimetric method[15], chemiluminescence method[16] and voltametric methods.[17] These
methods are required expensive or sophisticated instruments and not simple for routine
analysis. High performance liquid chromatography (HPLC) can be used for determination of
drugs and for purposes of control throughout the entire manufacturing process of drugs, as
well as quality control of the finished product. It has the advantages of being accurate,
sensitive, rapid, selective, and reproducible.[18,19] The present paper reports the development
of a new high performance liquid chromatography (HPLC) method for determination of
paracetamol in different type of pharmaceutical formulations and environmental water
samples.

MATERIALS AND METHODS

Apparatus
Chromatographic system consisted of an shimadzu HPLC model LC-20AT with UV detector
model SPD-20A and C18 supelco column (25cm ×4.6mm), 5µm particle size HPLC condition
were given in (Table.1).

Table (1): HPLC conditions.

Column Supelco C18 (25cm×4.6mm),5 µm
Wavelength 243 nm
Mobile phase Acetonitrile – pH3 (40:60)
Retention time 2.2 minutes
Flow rate 1.5 ml/min
Temperature Ambient
Injection volume 10 µL

Reagents
All chemicals used were of analytical or pharmaceutical grade and HPLC grade acetonitrile
were used throughout.

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Omar et al. World Journal of Pharmaceutical Research

Buffer solution (pH3)

This solution was prepared by dissolving 5.75 gm of monobasic ammonium phosphate in
about 80 ml of water, add sufficient acetic acid to adjust the pH to 3 and dilute to 100ml by
distilled water in a volumetric flask.[20]

A standard stock solution of paracetamol(1 mg/ml) was prepared in mobile phase. Working
standard solutions in a range of (10-100 µg/ml) were prepared by dilution from stock
solution.

HPLC method for determining paracetamol

A series of standard solution containing 10-100 µg/ml of paracetamol and the sample solution
of pharmaceutical preparations were applied respectively. 10µl aliquot of each solution was
injected into the column in a duplicate and the chromatograms were recorded. Calibration
graph was constructed by plotting the mean peak area versus concentration of paracetamol.
The concentration of the unknown was calculated from the regression equation derived from
the concentration and peak area data, or was read from calibration graph.

Procedures for pharmaceutical preparations

The recommended condition described above and mentioned in the HPLC method has been
applied satisfactorily for determination of paracetamol in different type of pharmaceutical
formulations provided from AL-Hokamaa Company for pharmaceutical industries (HPI)
Mosul-Iraq.

Tablets: Weigh and powder 20 tablets. Transfer an accurately weighed portion of the powder
equivalent to 5 mg paracetamol into 100 ml volumetric flasks and diluted with mobile phase
to the volume, and the amount of paracetamol was determined by comparing the peak area of
the assay preparation with the standard preparation at the same concentration.

Syrups and drops: Take a volume of syrups or drops containing 5 mg of paracetamol into
100 ml volumetric flasks and diluted with mobile phase to the volume, and the amount of
paracetamol was determined by comparing the peak area of the assay preparation with the
standard preparation at the same concentration.

Suppositories[21]: Tare a small dish and a glass rod, place in the dish NLT 5 Suppositories,
heat gently on a steam bath until melted, then stir, cool while stirring, and weigh. Transfer a
weighed portion of the mass, equivalent to 5 mg of paracetamol, to a separator. Add 30 ml of

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Omar et al. World Journal of Pharmaceutical Research

solvent hexane, and dissolve. Add 30 ml of water, shake gently, and allow the phases to
separate. Transfer the aqueous layer to a 100-mL volumetric flask, wash the solvent hexane
in the separator with three 30-mL portions of water, adding the washings to the volumetric
flask, and dilute with Mobile phase to volume. The amount of paracetamol was determined
by comparing the peak area of the assay preparation with the standard preparation at the same
concentration.

Procedure for industrial waste water: To demonstrate the practical applicability of the
proposed method, industrial waste water samples from al-hokamaa Company for drug
industries and medical appliances, Mosul-Iraq, were collected in polyethylene container
cleaned with nitric acid, and filtered through Whatman No.41 filter paper. Filtered samples
were stored at 4 C0 until analyzed which shows negative results, then the samples were spiked
with the concentrations ranging from 20-60 µg.ml-1 of paracetamol and then determined the
concentration of paracetamol as described under HPLC method for determining paracetamol.
Calculate the percentage recovery using a calibration graph previously prepared.

RESULTS AND DISCUSSION

The development of HPLC methods for the determination of drugs has received considerable
attention in recent years because of their importance in the quality control of drugs and
pharmaceutical products. The aim of this study was to develop a accurate, sensitive, rapid,
selective, and reproducible HPLC method for the determination of paracetamol in pure from,
its pharmaceutical formulations and industrial waste water samples using the most commonly
employed L1 column with UV detection. The detection wavelength of 243nm was chosen in
order to achieve a good sensitivity for quantitative determination of paracetamol in tablets,
syrups, drops, suppositories and wastewater samples. The mobile phase consisting of
acetonitrile : pH3 (40:60) offered a good separation at ambient temperature under these
conditions using a flow rate of 1.5 ml/min and retention time of 2.2 minutes as shown in the
chromatogram, Figure[2].

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Omar et al. World Journal of Pharmaceutical Research

Figure [2]: Typical chromatogram (paracetamole 50 µg/ml).

Under the described experimental conditions, the analytic peak was well defined and free
from tailing. paracetamol was determined by measuring the peak area. A plot of peak area
against concentration gave a linear relationship (r=0.999) over the concentration range 10-
100 μg /ml. Using regression analysis, the linear equation Y=3269x -203.8 was obtained
where Y is the mean peak area and X is the concentration in mg/ml figure 3.(Table.2) shows
some information about the calibration curve.

Figure [3]: Calibration curve for paracetamol.

Table (2): Some information about the calibration curve.

Linearity range Intercept Correlation coefficient Slope*
10-100 μg/ml 203.8 0.999 3269

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Omar et al. World Journal of Pharmaceutical Research

*
Slope: The ratio between the measured quantity in the analytical technique to the
concentration of the substance to be determined quantity to the small change in concentration
indicate a good sensitivity (high slope).[22]

Determination the limit of detection and limit of quantification (sensitivity)

The standard deviation at concentration zero was calculated and this value was used for the
calculation of the limit of detection and limit of quantification. The limits of detection (LOD)
and quantification (LOQ) were calculated using the following formulae: LOD= (3.3σ/s) and
LOQ=(10σ/s) where σ is the standard deviation of the response and s is the slope of the
regression line.[23] Limit of detection (LOD) and limit of quantification (LOQ) were found
3ng/ml and 9ng/ml respectively. The results indicate that the method was sensitive enough to
detect a concentration of 3ng/ml and able to quantify at a concentration of above 9ng/ml.

Precision and accuracy

The precision of the method was established by carrying out the analysis of paracetamol
(n=6) using the proposed method. The low value of standard deviation showed that the
method was precise. The results obtained are presented in (Table. 3). To ensure the reliability
and accuracy of the method recovery studies were carried out at five different levels. The
results of recovery studies were found to be accurate, mean recoveries being 100.51±1.09
(n=6) as shown in (Table.3).

Table (3): Method accuracy and precision.

Concentration of
RSD % Recovery %
paracetamol µg/ml
10 0.92 101.6
20 0.85 100.5
40 1.18 101.0
80 1.06 99.95
011 0.80 99.5
Mean(n=6) 0.962 100.51

Analytical application
The proposed method was successfully applied to the assay of paracetamol in pharmaceutical
formulations (tablets, syrups, drops and suppositories) and industrial waste water sample. The
result of analysis for pharmaceutical formulation (Table. 4) which reveals that there was close
agreement between the results obtained by the proposed method and label claim.

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 Omar et al. World Journal of Pharmaceutical Research

Table (4): Determination of paracetamol in pharmaceutical formulations.

Pharmaceutical formulations Label amount(mg) Found (mg) (Recovery %)
450 mg/tab 451.8 100.4
Colden tablet(HPI) 350mg/tab 349.65 99.9
Antispasmine tablet(HPI) 350mg/tab 349.58 99.88
Flu-out tablet(HPI) 350mg/tab 348.0 99.42
Algesic tablet(HPI) 500mg/tab 495.6 99.12
Paracetamol tablet(HPI) 325mg/tab 328.4 101.04
Paradin tablet(HPI) 120mg/5ml 119.0 99.16
Antipyrol syrup(HPI) 100mg/ml 99.3 99.3
Antipyrol drop(HPI) 120mg/5ml 122.2 101.83
Coldin syrup(HPI)
120mg/supp 121.1 100.91
Anti pyrol suppositories(HPI) 250mg/supp 248.5 99.4
500mg/supp 496.9 99.38

Mean of six determinations

The results of industrial waste water samples (Table. 5) show that the recovery values
obtained were closed to 100%.

Table (5): Determination of paracetamol in industrial waste water samples.

Paracetamol(µg /ml)
Water sample Recovery%
Taken Found
20 20.0 100
Industrial waste water 40 40.6 101.5
60 60.4 100.6
Mean of ten determinations.

Comparison of methods
The proposed method was compared with other reported HPLC methods. (Table. 6) shows
the comparison between the present method and other HPLC methods. The present method is
more applications than other reported HPLC methods.

Table (6): Comparison of the existing HPLC methods with the proposed method.
Parameters Method 1 Method 2 Method 3 Method 4
Ref 4 5 6 Proposed
Column C18 C18 C18 C18
Wavelength (nm) 042 240 001 243
Linear range µg/ml 10-100 μg/ml 5-120 μg/ml 011-0001 10-100 μg/ml
H2O- Acetonitrile
Methanol-
Mobile phase Acetonitrile – (pH2.5) Acetonitrile – pH 3.0 (40:60)
H2O(40:60)
(85:15) (15:85)
Retention time (minutes) 3.03 1.5 5.5 2.2
Flow rate 1.0 ml/min 1.0 ml/min 1.0 ml/min 1.5 ml/min
Tablets, Syrups, drops, Suppositories
Application Tablets Tablets Tablets
and industrial waste water

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Omar et al. World Journal of Pharmaceutical Research

CONCLUSION
In this study, accurate, simple, and rapid HPLC method was developed and validated for the
determination of paracetamol in pharmaceutical formulations and industrial waste water
samples. The method was selective using L1 analytical column and applicable to
pharmaceutical preparations. Thus the developed method was recommended for control
throughout the entire manufacturing process of drugs as well as quality control of the finished
product in view of its high recovery and precision.

ACKNOWLEDGMENTS
The first author [Dr. Nief Rahman Ahmad] wishes to express gratitude to the Al-hokamaa
state company of drug industries and medical appliance (HPI) Mosul– Iraq for providing gift
samples of paracetamol standard materials and pharmaceutical preparations and for
permission and facilities to carry out the research work.

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