Simultaneous RP-HPLC Determination of Diclofenac Potassium and Famotidine in Pharmaceutical Preparations
R.V Rele*, J.M. Parab, V.V. Mhatre and C.B. Warkar
D. G. Ruparel College, Matunga, Mumbai 400 016.
*Corresponding Author E-mail: warkarchandan@rediffmail.com
ABSTRACT:
A simple, fast and precise reverse phase high performance liquid chromatography method is developed for the simultaneous determination of famotidine and diclofenac potassium using methyl paraben as an internal standard. Chromatographic separation of these two drugs was performed on Inertsil C18 column ( 250 mm x 4.6 mm, 5 µm ) as stationary phase with a mobile phase comprising of 0.05 M KH2PO4 : methanol (35:65 v/v) at a flow rate of 1.5ml / min and UV detection at 254 nm. The retention time of famotidine, methyl paraben and diclofenac potassium were 1.95 min, 3.57 min and 12.66 min respectively. The proposed method was validated for linearity, accuracy, precision, limit of detection, limit of quantitation and solution stability. Linearity, accuracy and precision were found to be acceptable over the ranges of 100 – 300 µg / ml for famotidine and 250 – 750 µg / ml for diclofenac potassium. This method can be conveniently adopted for routine quality control analysis.
KEYWORDS: Famotidine, Diclofenac potassium, RP-HPLC
INTRODUCTION:
Famotidine, 2-[4-[2-(amino-sulfamoylimino-methyl) ethyl sulfanyl methyl]- 1,3-thiazol-2-yl] guanidine is used in the treatment of ulcers. Diclofenac potassium is chemically described as potassium-[(2,6-dichlorophenyl)amino]-phenyl acetate. It is a potassium salt of an aryl acetic acid derivative. It possesses analgesic, anti-inflammatory, and antipyretic activity.
Pharmaceutical preparation contains 20 mg of famotidine and 50 mg of diclofenac potassium. The drugs are official in U.S.P1, B.P2, I.P3. The literature survey revealed that no HPLC method was available for simultaneous determination of these two drugs in pharmaceutical preparations. Hence it is a novel HPLC method for determination of famotidine and diclofenac potassium from their combined dosage forms. The method described is simple, fast, precise and accurate for simultaneous determination of famotidine and diclofenac potassium from pharmaceutical preparation.
Famotidine (C8H15N7O2S3) 337.45 g/mol
Diclofenac potassium (C14H10Cl2KNO2) 334.24 g/mol
EXPERIMENTAL:
Materials:
Reference standards of famotidine and diclofenac potassium were obtained with certificate of analysis. The pharmaceutical formulation of combined dosage form of diclofenac potassium and famotidine tablets was procured from the market. Potassium dihydrogen orthophosphate and methanol were used of analytical grade from Qualigens. HPLC grade distilled water was obtained from millipore. Standard dilutions were prepared in diluent (methanol).
Instrumentation:
Chromatographic separation was preformed with Shimadzu LC 2010 high performance liquid chromatography system in isocratic mode, equipped with an auto sampler and a photo-diode array detector. Chromatograms and data were recorded by means of Class VP software.
Preparation of Standard Stock Solutions:
The stock solution of famotidine (2000 µg / ml) was prepared by dissolving 200.2 mg of famotidine (99.9 %) in 100 ml volumetric flask with diluent (solution A). The stock solution of diclofenac potassium (5000 µg / ml) was prepared by dissolving 500.4 mg of diclofenac potassium (99.6 %) in100 ml volumetric flask with diluent (solution B). Internal standard (methyl paraben) stock solution (1000 µg / ml) was prepared by dissolving 99.8 mg of methyl paraben in diluent in a 100 ml standard volumetric flask (solution C).
Working Standard Solution:
About 10.0 ml of each of the stock solutions A, B and C were transferred to a 100 ml volumetric flask and diluted up to the mark with diluent (methanol).
Sample Preparation:
Twenty tablets were weighed and their average weight was calculated. The tablets were crushed into a homogeneous powder and quantity equivalent to ten tablets was transferred in a 100 ml volumetric flask, dissolved in diluent and filtered through whatman no. 41 filter paper. A 10.0 ml of above filtrate was quantitatively transferred to a 100 ml volumetric flask, 10.0 ml of internal standard solution was added to it, and solution was diluted up to the mark with diluent.
Chromatographic conditions:
The criteria employed for selecting the mobile phase for the analysis of the drugs were cost involved, time required for the analysis and better separation of drugs. Chromatographic separation was performed on Inertsil C18 (250 mm x 4.6 mm, 5 µm particle) column. Different mobile phases were tried to develop for the analysis of famotidine and diclofenac potassium in their combined dosage form but the mobile phase which consisted of 0.05 M KH2PO4 in water: methanol (35:65 v/v) was suitable. The system was run at a flow rate of 1.5ml / min. About 20 µl of sample was injected in the chromatographic system and detection wavelength was set at 254 nm (figure 3) for simultaneous determination of famotidine and diclofenac potassium.
Method Development:
Different columns containing octyl and octadecyl silane stationary phase were tried for method development. Inertsil C18 column was found satisfactory over the other columns. The UV spectrum of famotidine and diclofenac potassium was scanned on photo diode array detector for selecting the optimum wavelength. Peak purity of famotidine and diclofenac potassium was checked using photo diode array detector. Wavelength 254 nm was considered satisfactory for detecting all the drugs with adequate sensitivity. A typical HPLC chromatogram for simultaneous determination of famotidine and diclofenac potassium from pharmaceutical formulation is shown in figure 1 and figure 2.
Fig. 1: Chromatogram of famotidine and diclofenac potassium with methyl paraben (internal standard) in standard preparation
Fig. 2: Chromatogram of famotidine and diclofenac potassium with methyl paraben (internal standard) in sample preparation
Figure 3: Overlain spectra of famotidine and diclofenac potassium
Results and Discussion:
System suitability:
System suitability tests were performed to confirm the suitability and reproducibility of the system. The test was carried out by injecting 20 µl standard solution of 200 µg / ml of famotidine and 500 µg / ml of diclofenac potassium using 100 µg / ml of methyl paraben as an internal standard in five replicates. The RSD values of famotidine and diclofenac potassium were 0.31 and 0.33 respectively. The RSD values were found to be satisfactory and meeting the all requirements (RSD less than 2.0 %). Theoretical plates, resolution, tailing factor were determined and are presented in table 1.
|
Parameters |
Famotidine |
Methyl paraben (IS) |
Diclofenac potassium |
|
Resolution |
- |
6.46 |
14.18 |
|
Tailing factor |
1.34 |
1.25 |
1.21 |
|
Theoretical plates |
3087 |
2962 |
2466 |
Linearity:
Linearity was evaluated by analysis of working standard solutions of famotidine and diclofenac potassium of seven different concentrations. The range of linearity was from 100 - 300 µg / ml for famotidine and 250 - 750 µg / ml for diclofenac potassium. The peak area ratio and concentration of each drug was subjected to regression analysis to calculate the calibration equations and correlation coefficients. The regression data obtained for famotidine and diclofenac potassium is represented in table 2. The result shows that with-in the concentration range mentioned above, there was an excellent correlation between peak area ratio and concentration.
Table 2. Results of Linearity
|
Analyte |
Slope |
Intercept |
Correlation coefficient (r2) (n=7) |
|
Famotidine |
15432.062 |
31252.52 |
0.9999 |
|
Diclofenac potassium |
24030.664 |
-29072.29 |
0.9997 |
Limit of detection and quantification:
The limit of detection (LOD) and limit of quantification (LOQ) were established at signal-to-noise ratio of 3:1 and 10:1 respectively. The LOD and LOQ of famotidine and diclofenac potassium were experimentally determined by injecting six injections of each drug. The LOD of famotidine and diclofenac potassium was found to be 0.2 µg / ml and 0.2 µg / ml respectively. The LOQ of famotidine and diclofenac potassium was found to be 0.5 µg / ml and 0.6 µg / ml respectively.
Precision:
Repeatability was studied by carrying out system precision. System precision was determined from results for six replicate injections of the mixed standard solution. The relative standard deviation was less than 2 %. Method precision was determined from the results of six independent determinations at 100 % of the test concentrations of famotidine and diclofenac potassium in the product. The % RSD was found to be 0.29 for famotidine and 0.15 for diclofenac potassium. The results obtained are tabulated in table 3.
Table 3. Results of Precision study
|
|
Famotidine |
Diclofenac potassium |
|
Drug found in mg/tab (mean) |
20.03 |
50.08 |
|
Mean % |
100.1 |
100.2 |
|
%RSD |
0.29 |
0.15 |
Accuracy:
To study accuracy of the method, recovery experiment was carried out by applying the standard addition method. A known quantity of drug substance corresponding to 100 %, 110 %, 120 % and 130 % of the label claim of drug were added, to determine if there are positive or negative interferences from excipients present in the formulation. Each set of addition were repeated three times. The accuracy was expressed as the percentage of analytes recovered by the assay. Table 4 lists the recoveries of the drug from a series of spiked concentrations. The results indicate the method is highly accurate for simultaneous determination of famotidine and diclofenac potassium.
Stability of Solution:
Stability of stock solutions of sample and standard containing 2000 µg / ml famotidine, 5000 µg / ml diclofenac potassium and 1000 µg / ml methyl paraben were checked for 24 hours at room temperature. The drug solutions were found to be stable for the specified period.
Method Application:
The validated high performance liquid chromatographic method was applied to simultaneous determination of famotidine and diclofenac potassium using methyl paraben as internal standard. Twenty tablets containing famotidine (20 mg /tab), diclofenac potassium (50 mg / tab) were crushed and used for sample preparation. The sample equivalent to ten tablets was weighed accurately and was dissolved in 80 ml diluent.
Table 4. Results of Accuracy experiment
|
Analyte |
Initial conc. (ppm) |
Conc. added (ppm) |
Total conc. (ppm) |
Conc. found (ppm) |
RSD (%) n= 3 |
Recovery (%) |
|
Famotidine |
200 200 200 |
20.0 40.0 60.0 |
220.0 240.0 260.0 |
219.83 243.61 259.06 |
0.50 0.57 0.39 |
99.9 101.5 99.6 |
|
Diclofenac potassium |
500 500 500 |
50.0 100.0 150.0 |
550.0 600.0 650.0 |
549.39 599.48 651.14 |
0.19 0.50 0.29 |
99.9 99.9 100.2 |
It was mixed well and further diluted to 100 ml with diluent. About 10.0 ml of above filtrate and 10.0 ml of internal standard solution were further diluted to get a solution of concentration of 200 µg / ml famotidine, 500 µg/ml diclofenac potassium and 100 µg / ml methyl paraben.
About 20 µl of this solution was injected into the chromatograph under the specified conditions. The analyte peaks were identified by comparison with observed retention times with those of respective standards. The peak areas obtained were used to calculate the drugs present.
conclusion:
Several mobile phases were tried but good peak shape and good resolution between famotidine, diclofenac potassium and methyl paraben were observed using the mobile phase mentioned in chromatographic conditions. The method after being completely validated showed satisfactory data for all the method validation parameters. The method was found to be specific. The low values of % RSD for method precision suggested that the method is precise. Linearity evaluated for the analyte peak showed a good linear response over a wide range of concentration. The linearity, precision, accuracy of the method proves that the method is specific, accurate, easily reproducible and can be used for simultaneous determination of famotidine and diclofenac potassium from pharmaceutical preparations.
ACKNOWLEDGEMENT:
Authors express sincere thanks to the principal of D. G. Ruparel College, Mumbai for guidance, encouragement and providing laboratory facilities.
REFERENCE:
1. United States Pharmacopoeia, US Pharmaceutical Convention Inc., Rockville, Volume I, II,
II. 2010 ; 3748.
2. British Pharmacopoeia, Her Majesty’s Stationary Office, London, Volume I, II, and III. 2010.
3. Indian Pharmacopoeia, Controller of Publication, Delhi, volume I, II, III. 2010 ; 2224.
Received on 23.01.2011 Modified on 14.02.2011
Accepted on 06.03.2011 © RJPT All right reserved
Research J. Pharm. and Tech. 4(4): April 2011; Page 638-641