Sunilkumar Adupa et al. / Journal of Pharmacy Research 2014,8(3),331-335 Research Article Available online through ISSN: 0974-6943 http://jprsolutions.info Simultaneous estimation of paracetamol caffiene and propyphenazone in bulk and pharmaceutical dosage form by RP-HPLC Sunilkumar Adupa1, Sravankumar Adupa2, Haribabu Simhadri3, Srikanth Varma Kalidindi4 1Department of Pharmaceutical Analysis & QA, Vikas College of Pharmaceutical Sciences, (V) Rayanigudem, (M) Suryapet, Nalgonda (District), A.P, 508376. 2St.peter’s Institute of Pharmaceutical Sciences, Vidyanagar, Hanamkonda, Warangal, A.P, 506001. 3Swamy Vivekananda College of Pharmacy, Tiruchengode , Namakkal Dist , T.N , 637205 4 JSSCollege of pharmacy, Ooty, Nilgiris (dist), T.N, 643001. Received on:21-01-2014; Revised on: 08-02-2014; Accepted on:05-03-2014 ABSTRACT A new, precise, rapid, accurate RP-HPLC method was developed for the estimation of Paracetamol Caffiene and Propyphenazone in bulk and pharmaceutical dosage form. After optimization the good chromatographic separation was achieved by Isocratic mode with a mixture of Methanol : Water (80:20) v/v and add 3ml Triethylamine to adjust the pH as the mobile phase with Thermohypersil C18 Octa Decyl Silane (250 x 4.6 mm, 5 µm), column as stationary phase at flow rate of 1.0 mL/min and detection wavelength of 243 nm. The Retention times for Paracetamol Caffiene and Propyphenazone were found to be 3.8, 4.7 and 5.7 min respectively. The linearity of this method was found in the concentration range of 50-150 µg/mL. The correlation coefficient R2 values of Paracetamol Caffiene and Propyphenazone found to be 0.997, 0.998, 0.998. The method of analysis was used for quantification of paracetamol, caffeine and propyphenazone pharmaceutical preparation with a co efficient of variation <2%.The % recovery of Paracetamol Caffiene and Propyphenazone were found to be in the range of 98-102% indicates that the proposed method is highly accurate. The specificity of the method shows good correlation between retention times of standard with the sample so, the method specifically determines the analyte in the sample without interference from excipients of formulation. The method was extensively validated according to ICH guidelines for Linearity, Range, Accuracy, Precision, specificity and Robustness. KEYWORDS: Caffiene, Isocratic, paracetamol, propyphenazone, RP-HPLC. INTRODUCTION Paracetamol is chemically N-(4-hydroxyphenyl)acetamide and the estimated by analytical methods such as few UV-Visible methods and structural formulais shown in Fig: 1.The molecular formula is C8H9NO2 high-performance liquid chromatographic (HPLC) method. The and molecular weight is 151.169g/mol. Caffeine is chemically 1,3,7- present developed method was simple, precise, specific and Trimethyl-1H-purine-2,6(3H, 7H)-dione 3,7-dihydro-1,3,7 trimethyl-1H- accurate [1-36]. purine-2, 6-dione and the structural formula is shown in Fig: 2. The molecular formula is C8H10N4O2 and molecular weight is 194.19g/mol. Propyphenazone is chemically 1,5 Dimethyl-2-phenyl-4-propan-2-yl- pyrazol-3-one and the structural formula is shown in Fig: 3. The mo- lecular formula is C14H18N2O and molecular weight is 230.306g/mol. These are stable at room temperature, freeely soluble in methanol and water. Paracetamol and propyphenazone are believed to exert their Fig. 1: Structural formula of Paracetamol. analgesic and antipyretic effect by inhibiting the cyclooxygenase (COX) and it is highly selective for COX-2. From the literature survey, it was found that paracetamol , propyphenazone and caffeine was *Corresponding author. Adupa Sunil kumar, M.Pharm., Pharmaceutical Analysis and Quality assurance, Vikas college of pharmaceutical sciences, (V)Rayanigudem-508376, Fig. 2: Structural formula of Caffeine. (M)Suryapet, Nalgonda (District), A.P,India. Journal of Pharmacy Research Vol.8 Issue 3.March 2014 331-335 Sunilkumar Adupa et al. / Journal of Pharmacy Research 2014,8(3),331-335 Preparation of Standard solution Take 2ml from the above solution in 50ml volumetric flask to this add 25ml of mobile phase. The volume was made up to mark with the mobile phase and sonicate it for 10min. The concentration of paracetamol , caffeine and propyphenazone was found 40 µg/ml. Preparation of Sample solution Take 20 tablets and crush the tablets after crushing take equivalent Fig. 3: Structural formula of Propyphenazone. weight of 50mg of paracetamol, 50mg of caffeine and 50mg of propyphenazone in 50ml volumetric flask to this add 25ml of mobile EXPERIMENTAL phase. The volume was made up to mark with the mobile phase and sonicate it for 10min. The concentration of paracetamol , caffeine and MATERIALS AND METHODS propyphenazone was found 1000 µg/ml. For HPLC, Waters 2690 series model equipped with Auto sample, PDA (2998) detector with Empower 2 software was employed for the Chromatographic condition investigation. All the chemicals used in the investigation were of The mobile phase containing water and methanol in the ratio of 80:20 HPLC grade. The chromatographic analysis was performed on a v/v was selected as the optimum composition of mobile phase, as this Themohypersil C-18 ODS column (150 x 4.6 mm, 5µm). The mobile solvent system resolved for the component ideally. The flow rate was phase consists of Methanol pH of 4.5 and Water in the ratio of 80:20 set to 1.0 ml/min and UV detection was carried out at 243 nm. The v/v. The optimized chromatographic conditions are summarized in mobile phase and sample was degassed by filtered through 0.4 µm Table 1. membrane filter paper. All determinations were performed at constant column temperature (350C). Table1: Optimized chromatographic conditions for the praposed method for Paracetamol Caffiene and Propyphenazone Preparation of Calibration curve and Analysis of Paracetamol, Caf- S.No Paremeter Optimized condition feine And Propyphenazone Appropriate aliquots were pipetted out from the standard stock solu- 1 Linear range (µg/ml) 50-150 tion (1000 µg/ml) into a series of 10 ml volumetric flasks. The volume 2 Detection wavelength (nm) 243 was made up to the mark with the mobile phase to get a set of solu- 3 Temparature (°C) 35 tions having the concentration, ranging from 50-150µg/ml of the drug. 4 Retention time of Paracetamol (Min) 3.883 5 Retension time of Caffeine (Min) 4.750 The solutions were injected using a 20µl fixed loop into the chromato- 6 Retension time of Propyphenazone (Min) 5.733 graphic system at the flow rate of 1.0 ml / min and the effluents were 7 LOD (PCM , CAF ,PROPY ) 0.0004,0.0002,0.0003 monitored at 243 nm, chromatograms were recorded. The paracetamol, 8 LOQ (PCM , CAF ,PROPY ) 0.001,0.005,0.004 caffeine and propyphenazone were eluted at 3.883, 4.750 and 5.733min as shown in Fig: 2. Table 2: System suitability test parameters for the praposed method for Paracetamol Caffiene and Propyphenazone S.No Paremeter Optimized condition 1 Retention time of Paracetamol (Min) 3.633 2 Retension time of Caffeine (Min) 4.483 3 Retension time of Propyphenazone (Min) 5.450 4 Theoritical plates 5552 Preparation of mobile phase: The mobile phase was prepared by mixing the Water and Methanol in the ratio of 80:20 , sonicate for 10min. After the solution was filtered through 0.45µm membrane filter to remove all fine particles and degas. Preparation of Standard stock solution Fig. 4: Chromatogram of Paracetamol, Caffeine and Propyphenazone Weighed accurately about 50 mg of standard Paracetamol , 50mg of by RP-HPLC method. standard Caffeine and 50mg of standard Propyphenazone and trans- ferred into a 50 ml volumetric flask to this add 25 ml of mobile phase. The calibration curve was constructed by plotting average peak area The volume was made up to mark with the mobile phase and sonicate versus concentration and was presented in Fig: 3. it for 10min. The concentration of paracetamol , caffeine and propyphenazone was found 1000 µg/ml. Journal of Pharmacy Research Vol.8 Issue 3.March 2014 331-335 Sunilkumar Adupa et al. / Journal of Pharmacy Research 2014,8(3),331-335 Linearity of Paracetamol Y= 64.14x + 29.792 Table4: Regression analysis of the calibration curve for the praposed R2 =0.9974 method for Caffeine. S.No Paremeter Optimized condition 1 Linearity (µg/ml) 50-150 2 Regression equation Y=mx+c 38.647x-89.394 3 Slope (m) 38.647 4 Intercept 89.394 5 Correlation coefficient (r2) 0.998 Area 6 Relative standard deviation 1.56 7 Retention time (min) 4.750 Table 5: Regression analysis of the calibration curve for the praposed method for Propyphenazone. Fig. 5: Calibration curve of Paracetamol at 243 nm by RP-HPLC S.No Paremeter Optimized condition method 1 Linearity (µg/ml) 50-150 2 Regression equation Y=mx+c 60.733x-285.75 3 Slope (m) 60.733 4 Intercept 285.75 5 Correlation coefficient (r2) 0.998 6 Relative standard deviation 1.67 7 Retention time (min) 5.733 Analysis of Paracetamol, Caffeine and Propyphenazone in Pharma- ceutical formulations The procedure for the preparation of the sample solution remains same as explained above. From this stock solution, various dilutions of the sample solution were prepared and analysed. A 20µl volume of each sample solution was injected into the sample injector of HPLC Fig. 6: Calibration curve of Caffeine at 243 nm by RP-HPLC method system and their chromatograms were recorded under the same chro- matographic conditions as described above. The area of each peak was determined at 243 nm and the amount of drug present in the sample was determined. Method validation The developed analytical method was subjected to validation with respect to various parameters such as accuracy, precision, linearity and range, robustness, ruggedness, LOD and LOQ as per the ICH guidelines. Table6: Summary of validation parameters for the for the praposed method for Paracetamol.