A Simple and Sensitive HPTLC Method for Estimation of ...

Asian J. Research Chem. 2(2): April- June., 2009Table: 1 Summary of validation parametersN0. Parameters Result1234567Linearity range (ng/ml)Correlation co-efficientPrecisionIntra day % CV (n = 3)Inter day % CV (n =3)Repeatability of sample application (n = 7)Repeatability of peak area ( n = 7)% RecoveryLimit of detectionLimit of quantificationSpecificity200-1200 ng/spot0. 99290.6 – 3.40.8 – 3.81.30.698.7 – 102.55 ng/spot20 ng/spotSpecificPreparation of standard solution of pioglitazone:Pioglitazone hydrochloride (10 mg) was weighedaccurately and transferred in 10 ml volumetric flask. Itwas dissolved in and diluted up to mark with methanol.The final solution contained 1000 µg of Pioglitazone perml of the solution (S1).The solution (0.5ml) was diluted further to 10 ml withthe same solvent. The final solution contained 50 µg ofpioglitazone per ml of the solution (S2).Preparation of sample solution:Ten tablets were weighed and finely powdered. Thepowder equivalent to Pioglitazone (10 mg) was weighedaccurately and mixed with methanol (5 ml) andsonicated for 10 minutes. The solution was filteredthrough Whatman NO. 41 filter paper. The residue waswashed thoroughly with methanol. The filtrate andwashings were combined in a 10 ml volumetric flask anddiluted to mark with methanol. The solution (0.5 ml)was further diluted to 10 ml with methanol.HPTLC method and chromatographic condition:The chromatographic estimations were performed usingfollowing conditions; stationary phase, precoated silicagel 60 F 254 aluminum sheets (20×10 cm) (pre-washedwith methanol. and dry in air); mobile phase,Toluene:methanol:ammonia (7:3:0.1 v/v); chambersaturation time, 30 min; Temperature, 29±4 o ; migrationdistance, 45 mm; wavelength of detection, 268 nm; slitdimensions, 3×0.3 mm; scanning speed, 5 mm/s.Following spotting parameters were used - band width,4 mm; space between two bands, 4 mm and sprayingrate, 10 sec/µl.Chromatographic separation:Twelve µl of standard or sample solution wasapplied on TLC plate under nitrogen stream usingsemiautomatic spotter. The plate was dried in airand developed up to 45 mm. at constant temperatureusing mixture of Toluene : methanol : ammonia (7: 3:0.1 v/v) as mobile phase in Camag twin-troughchamber previously saturated with mobile phase for30 min. The plate was removed from the chamber anddried. Photometric measurements were performed at268 nm in absorbance/reflectance mode with CamagTLC Scanner 3 with CATS4 software incorporatingthe track optimization option.Calibration curve of standard pioglitazone:Standard pioglitazone solution (4, 8, 12, 16, 20, and 24µl) was spotted on precoated TLC plate, usingsemiautomatic spotter under nitrogen stream. TheTLC plate was developed and photometricallyanalyzed as described under chromatographicseparation. The calibration curve was prepared byplotting peak area versus concentration (ng/spot)corresponding to each spot.Quantification of pioglitazone in tablet formulation:Twenty µl of sample solution ( 50 µg/ml ) wasapplied on prewashed TLC plate, developed andscanned as described in chromatographic separation.The amount of pioglitazone present in samplesolution was determined by fitting area values forpeak corresponding to pioglitazone into theequation of line representing calibration curve forpioglitazone.RESULTS AND DISCUSSION:In present work HPTLC method was developed forestimation of pioglitazone hydrochloride pure powderand its pharmaceutical formulation. HPTLC method iscost effective and less time consuming.Pioglitazone is soluble in methanol; therefore methanolwas selected as solvent. The formulation wasdissolved in methanol with sonication for 10 minto assure complete release of drug from theformulation matrix.Method optimization:For optimization, different mobile phases andcomposition were employed to achieve the goodseparation. The method development was initiated withusing a mobile phase of n-hexane –methanol in variousproportion. In the above conditions elution was verybroad for pioglitazone. Introduction of ethyl acetate inthe above mobile phase gave sharp peaks, but poorseparation and band broadening was observed. Earlyelution with a little separation was observed with themobile phase consisting of toluene-methanol (5:5). Inthe same mobile phase change proportion of toluene-208

methanol (7:3) gave reasonable Rf but not sharp band.Therefore need further optimization on the other hand,ammonia solution helped in sharpening of the peak.Finally, the mobile phase consisting of the mixture oftoluene: methanol: ammonia (7:3:0.1 v/v) could resolvepioglitazone spot with better peak shape. Combinationof toluene and methanol offered optimum migration(Rf= 0.50±0.03) and resolution of pioglitazone fromother components of formulation matrix. Even saturationof TLC chamber with mobile phase for 30 min assuredbetter reproducibility and better resolution. Pioglitazoneshows significant UV absorbance at wavelength 268 nm.Hence this wavelength has been chosen for detection inthe analysis of pioglitazone.The method was validated in terms of linearity, inter-dayand intra-day precision, repeatability of measurement ofpeak area as well as repeatability of sample application,accuracy and specificity. The limit of detection and limitof quantification were also determined.A representative calibration curve of pioglitazonewas obtained by plotting the mean peak area ofpioglitazone against the concentration over therange of 200 - 1200 ng/spot. A correlationcoefficient was found to be 0.9929 and RSD wasranging from 0.6- 3.4. The average linear regressionequation was represented as Y=2.5395X+1677.1, whereX=concentration of pioglitazone and Y=peak area. Thelimit of detection and limit of quantification forpioglitazone were found to be 5 ng/spot and 20 ng/spot,respectively.Inter-day and Intra-day variation range forpioglitazone was found to be 0.6 - 3.4 and 0.8 - 3.8respectively. Precision of the instrument was checked byrepeated scanning of the same spot (600 ng/spot) ofseven times without changing position of the plate and% CV for measurement of peak area was found to be0.64%. Repeatability of the method was checked byspotting 12 µl of standard solution seven times on TLCplate (n=7) and % CV for peak area was found to be1.3%. Both the % CV, for measurement of peak area andsample applications (less than 1% and 3%, respectively),ensuring proper functioning of HPTLC system.Accuracy of method was evaluated by calculatingrecovery of drug by standard addition method at 5 levelsof the calibration curve (n=3). The percentage recoverywas found to be 93.3 to 99.4% ensuring that the methodis accurate.The results indicate that the recovery of addedsample was between 99.6 - 102.5 %. This clearlyindicates that the method is accurate and precise.Asian J. Research Chem. 2(2): April- June., 2009three spectra indicated the purity of peak (correlation,r(S,M)=0.9999, r(M,E)=0.9993, fig. 2). The spectrum ofextracted from tablet was also compared with spectrumof standard, which showed correlation 0.9993. It wasobserved that the excipients present in formulationdid not interfere with the peak of pioglitazone.Different validation parameters for the proposed HPTLCmethod for determining pioglitazone content aresummarized in Table 1. This method was applied todetermine the content of pioglitazone in two differentmarket samples of single component pioglitazonetablets. The content and percentage of pioglitazone intwo different market samples were found to be 30.03mg, 100.1±2.9% and 29.82 mg, 99.4±1.5%, respectively(n=3). The results indicate that the proposed HPTLCmethod was found to be simple, specific, rapid, preciseand accurate for estimation of pioglitazone in itsformulations.CONCLUSION:The results indicate that the proposed method issimple, accurate, precise and specific, forestimation of pioglitazone in bulk and itsformulations.REFERENCES:1. http:// rxlist.com/actos-drug.htm.2. Sankar,D.G., Kumar, J.M.R., Reddy, M.V.V.N.Extractive spectrophotometric determination ofPioglitazone hydrochloride using both acidic and basicdyes. Asian Journal of Chemistry. 2004; 16 (1): 251-254.3. Radhakrishna, T., Sreenivas Rao, D., Om Reddy, G.Determination of pioglitazone hydrochloride in bulk andpharmaceutical formulations by HPLC and MEKCmethods. J. Pharm. Biomed. Anal. 2002; 29: 593.4. Zhong, W.Z., Williams, M.G. Simultaneous quantitationof pioglitazone and its metabolites in human serum byliquid chromatography and solid phase extraction. J.Pharm. Biomed. Anal. 1996; 14: 465.5. Zhong, W.Z., Lakings, D.B. Determination ofpioglitazone in dog serum using solid- phase extractionand high-performance liquid chromatography withultraviolet (229 nm) detection. J. Chromatogr. 1989; 30;490(2): 377.6. Sripalakit, P., Neamhom, P., Saraphanchotiwitthaya, A.High-performance liquid chromatographic method for thedetermination of pioglitazone in human plasma usingultraviolet detection and its application to apharmacokinetic study. J. Chromatogr. B: AnalyticalTechnologies in the Biomedical and Life Sciences. 2006;843 (2): 164-169.7. Lin, Z.J., Ji, W., Desai-Krieger, D., Shum, L.Simultaneous determination of pioglitazone and its twoactive metabolites in human plasma by LC-MS/MS. J.Pharm. Biomed. Anal. 2003; 33 (1): 101-108.The method is found to be specific for pioglitazone. Thepurity of the peak was determined by comparing thespectra at three different levels i.e. at peak start(S), peakapex (M) and peak end (E). Correlation between these209