RP-HPLC Method Development and Validation of Etodolac and Paracetamol in Combined Dosage Form
P. Balan*, I. Carolin Nimila, M. Lakshmi Prasanna, M. Vanaja Rani and S. Rajasekar
Dept. of Pharmaceutical Analysis, Faculty of Pharmacy, PRIST University, Thanjavur -614904, Tamilnadu, India.
*Corresponding Author E-mail: balannandu@gmail.com
ABSTRACT:
A simple, specific, accurate and precise reverse phase high pressure liquid chromatographic method has been developed for the simultaneous determination of Etodolac and Paracetamol from tablets. Stationary phase used was C18 column (symmetry C18, 250 mm x 4.5 mm). The sample was analyzed using acetonitrile: water in the ratio of 50:50 (pH adjusted to 5.8 with ortho phosphoric acid) as a mobile phase at a flow rate of 1.0 ml/min and detection was done at 232nm. The described method was linear over a concentration range of 2-14 μg/ml (r2 =0.999793 and0.999708) for Etodolac and Paracetamol respectively. The retention time for Etodolac and Paracetamol was found to be 1.932 and 2.456 min respectively, and recoveries from tablet were between 98.2%w/w and 101.2 %w/w. The limit of quantification (LOQ) for Etodolac and Paracetamol were found to be 0.15 and 0.04 μg/ml respectively The system suitability parameters were within the limit. The method developed was validated according to ICH guidelines which prove that the method was precise, accurate and can be used for routine determination of Etodolac and Paracetamol in bulk drug and its pharmaceutical dosage forms.
KEYWORDS: Etodolac, Paracetamol, RP-HPLC method, Validation.
INTRODUCTION:
Paracetamol is chemically N-(4-hydroxyphenyl) acetamide. Etodolac is chemically 1, 8 Diethyl-1, 3, 4, 9-tetrahydropyrano [3, 4-b] indole-1-acetic acid1. Both these drugs belong to class of nonsteroidal anti-inflammatory drugs (NSAIDs). A combination of 500 mg of Paracetamol and 400 mg of Etodolac is available commercially as tablet. This combination is used as analgesic an antipyretic. Paracetamol is official in Indian pharmacopeia2 and British pharmacopoeia B.P3. There are some works on spectrophotometric determination of Etodolac alone and its metabolites in biological samples4 .Simultaneous estimation of Etodolac and Aceclofenac5. Different chromatographic techniques were performed on Paracetamol with other combinations6-10 and till now no work on Etodolac and Paracetamol combination done. So we developed simple, rapid, accurate, reproducible and economical method for the simultaneous estimation of Etodolac and Paracetamol in tablet formulation and thus validated. The structures of Etodolac and Paracetamol were given in Fig 1 and Fig 2 respectively.
Fig 1: Structure of Etodolac
Fig 2: Structure of Paracetamol
MATERIALS AND METHODS:
Chemicals and Reagents:
Etodolac and Paracetamol were obtained from, IPCA Laboratories. Ltd. Mumbai. Water (HPLC grade), Acetonitrile (HPLC grade), orthophosphoric acid were of reagent grade. The pharmaceutical preparations of combination of Etodolac and paracetamol that is ETOVA-P tablet (IPCA Laboratories. Ltd. Mumbai).
Instrumentation
A Isocratic HPLC Waters 515 pump with Waters 2489 UV detector and RP-C18 column was used. A Rheodyne injector with a 20 μl loop volume was used for the injection of sample. The HPLC system was equipped with Empower software for data processing.
Chromatographic Conditions:
The mobile phase containing Acetonitrile: water (50:50) and the pH adjusted to 5.8 with orthophosphoric acid was found to resolve Etodolac and Paracetamol. The mobile phase was filtered on a 0.45 μ membrane filter and then ultrasonicated for 15 min. The flow rate was set to 1.0 ml/min. The 232.0 nm wavelength was selected for analysis. All determinations were performed at constant column temperature (25 ± 2ºC).
Preparation of Stock Solution:
8mg of Etodolac and 10mg of Paracetamol standard drugs were taken in 10ml volumetric flask and the volume was made up with mobile phase. Then 0.1ml of solution was taken and made up to 10ml so that final concentrations are 8 and 10 µg/ml.
Preparation of Sample Solution:
A total of 20 tablets were accurately weighed and triturated with a mortar and pestle. An amount equivalent to one tablet (containing 400 mg of Etodolac and 500mg of paracetamol) was transferred to a 50ml volumetric flask; 50 ml of mobile phase was added and the flask was kept in an ultrasonic bath for 15 min. The volume was made up to mark with mobile phase and the solution was filtered through Whatmann filter paper. The final volume of the solution was made up to 10 ml with mobile phase to get stock solutions containing 8μg/ml Etodolac and 10μg/ml Paracetamol. The diluted solution (10μg/ml) was analyzed under optimized chromatographic conditions and chromatogram is depicted. The results of analysis are given in Table1and the chromatogram given in Fig.3.
Table 1: Results of Analysis of tablet formulation
|
Parameters |
Etodolac |
Paracetamol |
|
%Estimated |
101% |
99.6% |
|
Standard deviation |
17327.24 |
13211.32 |
|
Limit of Detection (LOD) |
0.15µg/ml |
0.04µg/ml |
|
Limit of Quantitation (LOQ) |
0.4µg/ml |
0.14µg/ml |
|
%RSD |
0.14 |
1.80 |
Fig. 3: Chromatogram of Etodolac and Paracetamol.
Calibration Curve:
The linearity was observed at 2-14μg /ml range for both Etodolac and Paracetamol. The calibration curve was plotted according to their areas with respect to concentrations. Standard solutions were injected through 20 μl loop system and chromatograms were obtained using 1.0 ml/min flow rate. The effluent was monitored at 232.0nm. Calibration curve was constructed by plotting average peak area against concentration and regression equation was computed from Fig.4 and Fig.5. The system suitability parameters are given in Table.2.
Fig. 4: Calibration curve of Etodolac:
Fig. 5: Calibration Curve of Paracetamol:
Table 2: System suitability parameters:
|
Parameters |
Etodolac |
Paracetamol |
|
Linearity range |
2-14mcg/ml |
2-14mcg/ml |
|
Correlation coefficient |
0.999793 |
0.999708 |
|
Slope |
132841.3 |
73619.55 |
|
Retention time |
1.943min |
2.469min |
|
Resolution factor |
1.76 |
- |
|
USP plate count |
2165 |
3634 |
|
USP tailing |
1.09 |
1.26 |
Table 3: Results for Precision study:
|
Drug |
Intraday assay |
Inter day assay |
||
|
% Obtained |
% RSD |
% Obtained |
% RSD |
|
|
Etodolac |
101.2% |
0.154 |
101.1% |
0.142 |
|
Paracetamol |
98.2% |
1.62 |
99.6% |
1.80 |
Table 4: Recovery study data
|
Parameters |
Etodolac |
Paracetamol |
||
|
% Estimated |
% RSD |
% Estimated |
% RSD |
|
|
50% |
98.6% |
0.12 |
100.2% |
1.23 |
|
100% |
101% |
0.14 |
99.6% |
1.80 |
|
150% |
98.5% |
0.18 |
101% |
1.52 |
Table 5: Results of Robustness study
|
Factor |
Level |
Retention time |
%RSD |
||
|
Etodolac |
Paracetamol |
Etodolac |
Paracetamol |
||
|
pH of mobile phase |
7.6 |
2.082 |
2.502 |
0.497 |
0.495 |
|
8.0 |
2.050 |
2.509 |
0.09 |
0.07 |
|
|
Flow rate |
0.8 |
2.393 |
3.086 |
0.01 |
0.51 |
|
1.2 |
1.632 |
2.097 |
0.34 |
1.5 |
|
|
% of Acetonitrile |
48 |
2.102 |
2.508 |
0.88 |
0.27 |
|
52 |
1.987 |
2.455 |
0.13 |
0.29 |
|
Validation methods:
The developed method was validated in terms of linearity, accuracy, specificity, limit of detection, limit of quantification, intra-day and inter-day precision and repeatability of measurement.
Specificity:
The peak purity of Etodolac and paracetamol were assessed by comparing the retention time (Rt) of standard Etodolac and paracetamol. Good correlation was obtained between the retention time of standard and sample of Etodolac and paracetamol.
Precision:
Precision was evaluated by carrying out six independent sample preparation of a single lot of formulation. The sample solution was prepared in the same manner as described in sample preparation. Percentage relative standard deviation (%RSD) was found to be less than 2% for within a day and day to day variations, which proves that method is precise. The results for precision are given in Table 3.
Accuracy (Recovery studies)
To check the degree of accuracy of the method, recovery studies were performed in triplicate by standard addition method at 50%, 100% and 150%.Known amounts of standard mixture of Etodolac and paracetamol was added to pre-analyzed samples and was subjected to the proposed HPLC method. Results of recovery studies are shown in Table 4.
Robustness of method:
To evaluate the robustness of the developed RP-HPLC small deliberate variations in the optimized method parameters were done. The effect of change in flow rate, pH and mobile phase ratio on the retention time and tailing factor were studied. The method was found to be unaffected by small changes like ±0. 2 change in pH, ±0. 2 change in flow rate and ±2change in mobile phase. The results of robustness are tabulated in Table. 5.
Limit of Detection (LOD) and Limit of Quantitation (LOQ):
The LOD and LOQ of the developed method were determined by analyzing progressively low concentration of the standard solutions using the developed methods. The LOD is the smallest concentration of the analyte that gives a measurable response (signal to noise ratio of 3). The LOQ is the smallest concentration of the analyte, which gives response that can be accurately quantified (signal to noise ratio of 10).
RESULTS AND DISCUSSION:
The regression value was found to be 0.99979 and 0.99970 for Etodolac and Paracetamol respectively, which shows the response, is linear from 2µg- 14μg /ml respectively. Selectivity experiment showed that there is no interference or overlapping of the peaks either due to excipients or diluents with the main peak of Etodolac and Paracetamol. The percentage RSD for precision is <2 which confirms that method is sufficiently precise and the total run time required for the method is only 5mins for eluting both Etodolac and Paracetamol. The proposed method is simple, fast, accurate, and precise and can be used for routine analysis in quality control of Etodolac and Paracetamol, different mobile phases were tried and the proposed chromatographic conditions were found to be appropriate for the quantitative determination. The developed method was also validated and results were found to be within the limit.
CONCLUSION:
The most striking feature of this method is its simplicity and rapidity, non- requiring- consuming sample preparations such as extraction of solvents, heating, degassing which are needed for HPLC procedure. This method can be employed for routine quality control analysis. The described method gives accurate and precise results for determination of Etodolac and paracetamol mixture in marketed formulation.
ACKNOWLEDGEMENT:
The authors are thankful to PRIST University, Thanjavur, Tamilnadu for providing chemicals and technical support to perform the work done. The authors are also thankful to IPCA Laboratories, Mumbai, and Maharashtra, India for providing the gift samples of Paracetamol and Etodolac.
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3. British Pharmacopoeia. Vol. II, London: Her Majesty's Stationary Office; 1998. p.1854.
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Received on 04.04.2011 Modified on 02.05.2011
Accepted on 12.05.2011 © AJRC All right reserved
Asian J. Research Chem. 4(7): July, 2011; Page 1073-1076