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ISSN: 0973-4945; CODEN ECJHAO
E-Journal of Chemistry
http://www.ejchem.net 2012, 9(3), 1347-1356
Stability-Indicating RP-HPLC Method for Analysis of
Paracetamol and Tramadol in a Pharmaceutical
Dosage Form
RAJESH M. KAMBLE* AND SHRAWAN G. SINGH
Department of Chemistry, University of Mumbai, Vidyanagari,
Santacruz (East), Mumbai- 400 098, (MS), India
chemrajkam@yahoo.co.in
Received 19 October 2011; Accepted 30 December 2011
Abstract: A simple, isocratic, rapid and accurate reversed phase high
performance liquid chromatography method was developed for the quantitative
determination of paracetamol and tramadol in commercial medicinal tablets.
The chromatographic separation was achieved on an Inertsil C18 (250 mm x 4.6
mm, 5µm) column using water pH 3.4 with orthophosphoric acid: methanol
(60:40, v/v) as a mobile phase, and UV detection at 228 nm. The
chromatographic resolutions between paracetamol and tramadol were found
greater than five. The linear range for paracetamol and tramadol were
20.8‒39.0 µg/ml and 2.4‒4.5 µg/ ml was obtained with correlation
coefficients ≥0.999 for each analyte. The retention time were found to be 2.1
and 3.9 min for tramadol and paracetamol respectively. Paracetamol and
tramadol was subjected to stress conditions (hydrolysis (acid, base) oxidation,
photolysis and thermal degradation) and the stressed samples were analyzed by
use of the method. The major degradation was observed in acid and minor in
base, thermal, oxidation and photolysis. The forced degradation studies prove
the stability indicating power of the method.
Keywords: Paracetamol, Tramadol, RP-HPLC, Validation, Stress studies.
Introduction
In recent times there has been an increased tendency toward development of stability-
indicating assays1‒3, using the approach to stress testing enshrined in International
conference on harmonization (ICH) guideline Q1A (R2)4. This approach is being extended
to drug combinations to enable accurate and precise quantification of several drugs in the
presence of their degradation products. Paracetamol is chemically 4-hydroxy acetanilide
(Figure 1). It is a weak inhibitor of peripheral cyclooxygenase and its analgesic effects may
arise from inhibition of prostanoid synthesis in the CNS. The antipyretic effects of
paracetamol are due to its action at the level of the hypothalamus to reduce pyrogen-initiated
RAJESH M. KAMBLE
1348
alterations in body temperature by inhibiting prostaglandin synthesis56. Tramadol
hydrochloric (±)-cis-2-(dimethylamino) methyl-1-(3-methoxy-phenyl) cyclohexanol
hydrochloride (Figure 1), a synthetic analogue of codeine, is a centrally acting analgesic
agent7. It has been used since 1977 for the relief of severe physical pain and has been the
most widely sold opioid analgesic drug in the world8. There are many reported methods for
analysis of tramadol913 or paracetamol1417 either alone or in combination with other
drugs1820 in pharmaceutical dosage forms. Very few reports are there on simultaneous
estimation of paracetamol and tramadol. They were determined in human plasma samples
using liquid chromatography (LC-MS)2122. In tablets they were estimated using
spectrophotometry2324, HPTLC2526, GC-MS27 and HPLC2730 methods. Till date, to the best
of our knowledge, no method has been reported in the literature for the stability indicating
assay for these two compounds simultaneously. This manuscript describes the development
and validation, in accordance with ICH guidelines, of rapid, economical, precise and
accurate stability-indicating isocratic reversed-phase HPLC method for analysis of
paracetamol and tramadol in the presence of its degradation products.
Paracetamol Tramadol HCl
Figure 1. Structure of paracetamol and tramadol.
Experimental
Chemicals and reagents
Paracetamol and tramadol reference standards were obtained from sigma Aldrich,
Acetonitrile, methanol, water (HPLC grade), orthophosphoric acid (AR) were obtained from
Merck Ltd.
Stock solutions of paracetamol (65 mg) and tramadol (7.5 mg) were weighed
independently and transferred separately to 100 ml volumetric flask. The drug was dissolved
in methanol and dilution was made to the mark. A mixed standard solution was prepared
from above solution by dissolving 4.0 ml of both stock solution in to 100 ml volumetric
flask and diluted up to the volume with mobile phase to get final concentration of 26.0
µg/ml of paracetamol and 3.0 µg/ml of tramadol.
Instrumentation and Chromatographic conditions
An isocratic HPLC (Thermo Electron Corporation) with pump P-2000, UV/VIS-1000 was
used. The analysis was carried out on Inertsil ODS C18 (250 mm x 4.6 mm, 5 µm), a manual
injector with a 20 µl loop was used for the injection of sample solution and the mobile
phase.
Stability-Indicating RP-HPLC Method for Analysis of Paracetamol
1349
Freshly prepared water adjusted pH 3.4 with orthophosphoric acid and methanol in the
ratio of 60:40 v/v was used as mobile phase. These were filtered through 0.45 µm membrane
filter and sonicated before use. The flow rate of mobile phase was 1.0 ml/min. The column
was maintained at ambient temperature. The detection was carried out by at 228 nm and
runtime was around 8 min.
Preparation of tablet solution
Twenty tablets were accurately weighed and finely powdered. A quantity of powder
equivalent to 65 mg of paracetamol and 7.5 mg of tramadol were transferred to 100 ml
volumetric flask and dissolved in methanol. The solution was filtered through Whatmann
filter paper. From the above solution 4.0 ml was taken and diluted to 100 ml with mobile
phase to give test solution containing 26.0 µg/ml of paracetamol and 3.0 µg/ml of tramadol.
These sample solution were injected six different times.
Results and Discussion
Optimization of chromatographic conditions
The primary objective in developing the present stability-indicating HPLC method was to
achieve resolution between paracetamol and tramadol and its degradation products. The
choice of chromatographic conditions selected was based on symmetry of peak shape and
reduction of chromatographic analysis. The mobile phase consisting of buffer (water
adjusted to pH 3.4 with orthophosphoric acid) and methanol in composition of (60:40 v/v)
was selected. In mobile phase organic solvent was selected because of its favourable UV
transmittance, low viscosity and provides good resolution between two drugs. The water
consisting of buffer helps in obtaining sharp peaks and produces good resolution with
retention time 2.1 and 3.9 min for tramadol and paracetamol respectively. The detection was
carried at 228 nm in UV where both drugs showed good absorbance. The chromatographic
analysis time was less than 8.0 min. Resolution of the two components was around 6.0 with
clear baseline separation was obtained as shown in Figure 2.
Figure 2. Typical chromatogram obtained from tramadol and paracetamol solution.
Forced Degradation Studies
In true sense, ICH guideline Q1AR and the ICH’s Common Technical Document31 suggest
stress testing only of the drug substance. For drug products, however, a definition and
guidelines of ‘Stress Testing (drug product)’ is provided in Q1AR32. In literature there exist
RAJESH M. KAMBLE
1350
many reports where stability-indicating assay has been established by carrying out stress
tests directly on pharmaceutical formulations33‒38.
In the present study the stress testing was carried out on paracetamol and tramadol
containing pharmaceutical formulation and the degradation was observed when the analyte
was subjected to acid, base, oxidation, photolytic and thermal stress conditions. The drug
substances paracetamol and tramadol under acidic conditions leads to formation of an
unknown degradation peaks at the retention time 4.8 and 6.5 min and in oxidative condition
at 7.8 min. Intentional degradation was attempted to stress conditions of acidic (using 0.1N
HCl), basic degradation (using 0.1N NaOH), oxidative degradation (using 3.0% H2O2),
photolytic degradation (UV degradation) and thermal degradation (heated at 100° C) and to
evaluate the ability of the proposed method to separate paracetamol, tramadol and its
degradation products. Concentration of Paracetamol and tramadol (26.0 µg/ml and 3.0
µg/ml) was used in all the degradation studies. After completion of the degradation
processes, the solutions were neutralized and diluted with mobile phase.
Forced degradation in acidic media was performed by taking 4.0 ml aliquot from stock
solution in 50 ml of round bottom flask, to this 5.0 ml of 0.1 N HCl was added and the
solution was refluxed for 12 hrs and neutralized using same strength of 0.1 N NaOH.
Appropriate aliquot was taken from the above solution and diluted with mobile phase to a
final concentration of 26.0 and 3.0 µg/ml of paracetamol and tramadol.
Forced degradation in basic media was performed by taking 4.0 ml aliquot from stock
solution in 50 ml of round bottom flask, to this 5.0 ml of 0.1 N NaOH added and the solution
was refluxed for 12 hrs and neutralized using same strength of 0.1 N HCl. Appropriate
aliquot was taken from the above solution and diluted with mobile phase to a final
concentration of 26.0 and 3.0 µg/ml of paracetamol and tramadol.
Oxidative degradation was performed by taking 4.0 ml aliquot from stock solution in 50
ml of round bottom flask, to this 5.0 ml of 3% v/v of hydrogen peroxide added and the
solution was refluxed for 12 hrs. The solution was diluted with mobile phase to obtain a
final concentration of 26.0 and 3.0 µg/ml of paracetamol and tramadol.
To study the effect of UV light, the drug powder was exposed to light. Approximately 25
mg of tablet powder was spread on a glass dish in a layer that was less than 2 mm thick and
was prepared in mobile phase. All samples for photostability testing were placed in a UV
light cabinet and which were exposed for 12 hrs resulting in an overall illumination of ≥
210Wh/m2 at 25° C with UV radiation. Following removal of glass dish from the UV light
cabinet, all samples were prepared for analysis as previously described.
To study the effect of temperature, the tablet powder was exposed to dry heat in a
controlled temperature oven at 100° C for 12 hrs.
Stress Degradation of Formulation
Chromatograms obtained from paracetamol and tramadol tablet solution after degradation
under acidic, basic, oxidizing, photolytic and thermal conditions are shown in Figures 3‒7.
In acid (0.1 N hydrochloric acid) degradation study showed 14.52% and 32.82%
degradation for tramadol and paracetamol respectively at reflux temperature after 12 hrs. A
major degradation product was observed at 4.8 and 6.5 min.
Stability-Indicating RP-HPLC Method for Analysis of Paracetamol
1351
The degradation in base (0.1 N sodium hydroxide) was found to be 4.75% and 6.19% for
tramadol and paracetamol respectively at reflux temperature after 12 hrs. No degradation
product was observed.
Oxidative degradation study in 3% hydrogen peroxide gave around 4.74% and 1.15%
degradants at reflux temperature at end of 12 hrs. A degradation product was observed at 7.8
min.
In photolytic, UV degradation the drug degraded was 2.24% and 0.65% of tramadol and
paracetamol for 12 hrs. No degradation peak was obtained.
The thermal study was carried out by exposure of tablet powder to dry heat at 100° C for
12 hrs. There were no degradation peak observed in chromatogram and degradation was
6.50% and 1.68% for tramadol and paracetamol respectively.
The results from force degradation studies are summarized in Table 1.
Figure 3. Representative chromatogram obtained from Acidic degradation (0.1 N HCl).
Figure 4. Representative chromatogram obtained from Basic degradation (0.1 N NaOH).
RAJESH M. KAMBLE
1352
Figure 5. Representative chromatogram obtained from oxidative degradation (3% H2O2).
Figure 6. Representative chromatogram obtained from UV radiation in UV light cabinet.
Figure 7. Representative chromatogram obtained from thermal degradation at 100°C.
Method Validation
The test method was validated for specificity, linearity, precision, accuracy, range, stability
of sample solution and robustness were found to be meeting the predetermined acceptance
criteria. The validated method was found to be specific, linear, precise, accurate and robust
for the assay of paracetamol and tramadol in their tablet dose form. Hence, this method can
be introduced into the use for the assay of paracetamol and tramadol in tablets.
Stability-Indicating RP-HPLC Method for Analysis of Paracetamol
1353
Table 1. Results from analysis of samples from the forced degradation study.
Precision, as % RSD evaluated from the area of paracetamol and tramadol peaks from
six replicated injections of standard solution indicating that the measurement system was
precise for the determination of paracetamol and tramadol in tablets. Also, % RSD of
intraday and interday precisions of the proposed method was determined by estimating the
corresponding responses 6 times on the same day and 6 on another day for same
concentration of paracetamol (26.0 µg/ml) and tramadol (3.0µg/ml). The % RSD for both
intraday and interday was not more than 2.0%.
The accuracy of the method was studied by measuring of recovery after standard addition
method of paracetamol and tramadol at 80, 100, 120 and 150% level. Known amounts of
standard solutions of paracetamol (20.8, 26.0, 31.2 and 39.0 µg/ml) and tramadol (2.4, 3.0,
3.6 and 4.5 µg/ml) were spiked to prequantified sample solutions. The percent recoveries
obtained were between 98.0 to 102% for both samples. These high values indicate that the
method is accurate.
Linearity, the value of correlation coefficient obtained from the linearity studies
(correlation coefficient for paracetamol was 0.99986 and for Tramadol was 0.99974)
indicating that the response was linear for paracetamol and Tramadol peaks over the
concentration range from 20.839.0 µg/ml for paracetamol and 2.44.5 µg/ml for Tramadol.
A calibration curve for each sample was obtained by plotting area response versus
concentration which gave a straight line corresponding to the equation: y = mx + c. The
results showed that good correlation existed between the park area and concentration of the
analytes.
Stress Time Tramadol Paracetamol Remarks
condition
Ret.
time
%
RS
D
%
Degrd.
Ret.
time
%
RS
D
%
Degrd.
Acid
degradation
12 hrs
1.95
0.41
14.52
4.03
0.62
32.82
Significant
degradation
product found at
4.8 and 6.5 min
Basic
degradation
12 hrs
2.07
0.79
4.75
4.06
0.35
6.19
No degradation
product formed
Oxidative
degradation
12 hrs
2.18
0.78
4.74
4.00
0.48
1.15
Significant
degradation
product found at
7.8 min
UV
degradation
12 hrs
2.20
0.76
2.24
3.90
0.67
0.65
No degradation
product formed
Thermal
degradation
12 hrs
1.88
0.32
6.50
4.01
0.39
1.68
No degradation
product formed
RAJESH M. KAMBLE
1354
In the system suitability test of solution containing 26.0 µg/ml of paracetamol and 3.0
µg/ml of tramadol were prepared and injected six times (n=6). The standard and sample
solutions were stored at room temperature and analyzed over the time period of 0 hrs, 12 hrs
and 24 hrs. Then different parameters such as retention time, theoretical plates, tailing factor
and resolution were calculated from the obtained chromatograms. Thus absolute difference
between % assay values was not more than ± 2.0 % compared to the initial values which
were in agreement with the USP requirements. Results from validation are presented in
Table 2.
Table 2. Results from Validation of method.
Parameter
Paracetamol
Tramadol
Linearity coefficient
0.99974
0.99986
Precision %RSD
0.16
0.19
Intermediate Precision % RSD
0.20
0.25
Solution stability % RSD 12 hrs
% RSD 24 hrs
0.25
0.11
0.17
0.36
Retention times Minutes
3.9
2.1
Resolution
6.07
---
Table 3. Results from robustness testing.
Chromatographic
change factor
Variation
Paracetamol
Tramadol
Ret.
time
(%)
RSD
Ret.
time
(%)
RSD
Flow rate (ml/min)
0.8
-0.2
4.82
0.42
2.68
0.28
1.0
0.0
3.90
0.10
2.19
0.14
1.2
+0.2
3.23
0.43
1.79
0.41
Wavelength (nm)
226
-0.2
3.88
0.38
2.16
0.33
228
0.0
3.89
0.13
2.18
0.16
230
+0.2
3.60
0.36
2.14
0.32
Mobile phase
38:62
-0.2
4.01
0.50
2.23
0.31
(Methanol: Buffer)
40:60
0.0
3.89
0.19
2.19
0.28
42:58
+0.2
3.74
0.32
2.09
0.81
Stability-Indicating RP-HPLC Method for Analysis of Paracetamol
1355
As recommended in the ICH guidelines, a robustness assessment was performed during
the development of the analytical procedure. The robustness of the method was investigated
under a variety of conditions including slightly changes of flow rate (1.0 ± 0.2 ml/min),
change in detection wavelength ( 228 ± 2.0 nm), change in composition of mobile phase
methanol : buffer (38:62, 40:60, 42:58). One factor at a time was changed to estimate the
effect. The degree of reproducibility of the results obtained as a result of small deliberate
variations in the method parameters has proven that the method is robust and the data is
summarized in Table 3.
Conclusion
The method developed for quantitative analysis of paracetamol and tramadol is rapid,
accurate, precise, selective and reproducible. The method was completely validated and
satisfactory results were obtained for all the characteristics tested. The major degradation
was obtained in acidic and minor in basic, thermal, oxidative and photolytic stress
conditions. The forced degradation studies prove the stability indicating power of the
method and can be used to assess the stability of paracetamol and tramadol in the bulk drug
and in pharmaceutical dosage forms. The method can be conveniently used for assay of the
pharmaceutical dosage forms containing paracetamol and tramadol in quality control
laboratory.
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