INTRODUCTION:

Pioglitazone is an oral antidiabetic agent belonging to the class of thiazolidinediones that acts primarily by decreasing insulin resistance. It is used in the management of type 2 diabetes mellitus. It improves sensitivity to insulin in muscle and adipose tissue and inhibits hepatic gluconeogenesis also improves glycemic control while reducing circulating insulin levels. Pioglitazone [(±)-5-[[4-[2-(5-ethyl-2- pyridinyl) ethoxy]phenyl]methyl]-2,4-] thiazolidinedione mono-hydrochloride belongs to a different chemical class and has a different pharmacological action than the sulfonylureas, metformin, or the α-glucosidase inhibitors (1).

Determination of pioglitazone by various analytical methods like spectrophtometric method² and HPLC and MECK method³ in tablet dosage form, HPLC and solid phase extraction method in human serum⁴ and in dog serum⁵, HPLC and LC MS in human plasma^{6, 7} have been reported. But these methods are sophisticated, expensive and time consuming when compared to simple HPTLC method. Pioglitazone is not official in any pharmacopoeia. There is a need for a simple, rapid, cost effective and reproducible method for assay of pioglitazone in its dosage forms.

Therefore, it was thought of interest to develop simple, speedy, accurate and cost effective method for the analysis of pioglitazone in its tablet formulation.

This paper describes development and validation of simple, specific, sensitive, accurate and precise HPTLC method for the estimation of pioglitazone in bulk and its formulation.

MATERIALS AND METHODS:

Materials:

Pioglitazone (Hydrochloride salt) working standard was procured as a gift sample from Torrent Pharma. Ltd., Ahmedabad. Silica gel 60 F₂₅₄ TLC plates (20×20 cm, layer thickness 0.2 mm, E. Merck, Germany) were used as stationary phase. Two single component uncoated tablet formulations of Pioglitazone (30 mg) (formulation A- Gatilox tablets, manufactured by Sun Pharma Limited, Baroda and formulation B - Tequin tablets, manufactured by Cadila Healthcare Limited, Ahmedabad) were purchased from market. Toluene, ammonia (SD’S) and methanol (A.R., Ranbaxy Ltd., New Delhi) were used for mobile phase preparation and as solvents.

A Camag HPTLC system (Switzerland) comprising of Camag Linomat IV semiautomatic sample applicator, Camag TLC Scanner 3, Camag twin-trough chamber (10×10 cm), Camag CATS 4 software, Hamilton syringe (100 µl), Shimadzu libror AEG- 220 weighing balance, Sonicator (Frontline Fs-4, Mumbai) were used during the study.

Preparation of standard solution of pioglitazone:

Pioglitazone hydrochloride (10 mg) was weighed accurately and transferred in 10 ml volumetric flask. It was dissolved in and diluted up to mark with methanol. The final solution contained 1000 µg of Pioglitazone per ml of the solution (S1).

The solution (0.5ml) was diluted further to 10 ml with the same solvent. The final solution contained 50 µg of pioglitazone per ml of the solution (S2).

Preparation of sample solution:

Ten tablets were weighed and finely powdered. The powder equivalent to Pioglitazone (10 mg) was weighed accurately and mixed with methanol (5 ml) and sonicated for 10 minutes. The solution was filtered through Whatman NO. 41 filter paper. The residue was washed thoroughly with methanol. The filtrate and washings were combined in a 10 ml volumetric flask and diluted 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 using following conditions; stationary phase, precoated silica gel 60 F₂₅₄ aluminum sheets (20×10 cm) (pre-washed with methanol. and dry in air); mobile phase, Toluene:methanol:ammonia (7:3:0.1 v/v); chamber saturation time, 30 min; Temperature, 29±4^o; migration distance, 45 mm; wavelength of detection, 268 nm; slit dimensions, 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 spraying rate, 10 sec/µl.

Chromatographic separation:

Twelve µl of standard or sample solution was applied on TLC plate under nitrogen stream using semiautomatic spotter. The plate was dried in air and developed up to 45 mm. at constant temperature using mixture of Toluene : methanol : ammonia (7: 3: 0.1 v/v) as mobile phase in Camag twin-trough chamber previously saturated with mobile phase for 30 min. The plate was removed from the chamber and dried. Photometric measurements were performed at 268 nm in absorbance/reflectance mode with Camag

TLC Scanner 3 with CATS4 software incorporating the track optimization option.

Calibration curve of standard pioglitazone:

Standard pioglitazone solution (4, 8, 12, 16, 20, and 24 µl)

was spotted on precoated TLC plate, using semiautomatic spotter under nitrogen stream. The TLC plate was developed and photometrically analyzed as described under chromatographic separation. The calibration curve was prepared by plotting peak area versus concentration (ng/spot) corresponding to each spot.

Quantification of pioglitazone in tablet formulation:

Twenty µl of sample solution ( 50 µg/ml ) was applied on prewashed TLC plate, developed and scanned as described in chromatographic separation. The amount of pioglitazone present in sample solution was determined by fitting area values for peak corresponding to pioglitazone into the equation of line representing calibration curve for pioglitazone.

RESULTS AND DISCUSSION:

In present work HPTLC method was developed for estimation of pioglitazone hydrochloride pure powder and its pharmaceutical formulation. HPTLC method is cost effective and less time consuming.

Pioglitazone is soluble in methanol; therefore methanol was selected as solvent.

The formulation was dissolved in methanol with sonication for 10 min to assure complete release of drug from the formulation matrix.

Method optimization:

For optimization, different mobile phases and composition were employed to achieve the good separation. The method development was initiated with using a mobile phase of n-hexane –methanol in various proportion. In the above conditions elution was very broad for pioglitazone. Introduction of ethyl acetate in the above mobile phase gave sharp peaks, but poor separation and band broadening was observed. Early elution with a little separation was observed with the mobile phase consisting of toluene-methanol (5:5). In the same mobile phase change proportion of toluene-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 of toluene: methanol: ammonia (7:3:0.1 v/v) could resolve pioglitazone spot with better peak shape. Combination of toluene and methanol offered optimum migration (Rf= 0.50±0.03) and resolution of pioglitazone from other components of formulation matrix. Even saturation of TLC chamber with mobile phase for 30 min assured better reproducibility and better resolution. Pioglitazone shows significant UV absorbance at wavelength 268 nm. Hence this wavelength has been chosen for detection in the analysis of pioglitazone.

The method was validated in terms of linearity, inter-day and intra-day precision, repeatability of measurement of peak area as well as repeatability of sample application, accuracy and specificity. The limit of detection and limit of quantification were also determined.

A representative calibration curve of pioglitazone was obtained by plotting the mean peak area of pioglitazone against the concentration over the range of 200 - 1200 ng/spot. A correlation coefficient was found to be 0.9929 and RSD was ranging from 0.6- 3.4. The average linear regression equation was represented as Y=2.5395X+1677.1, where X=concentration of pioglitazone and Y=peak area. The limit of detection and limit of quantification for pioglitazone were found to be 5 ng/spot and 20 ng/spot, respectively.

Inter-day and Intra-day variation range for pioglitazone was found to be 0.6 - 3.4 and 0.8 - 3.8 respectively. Precision of the instrument was checked by repeated scanning of the same spot (600 ng/spot) of seven times without changing position of the plate and % CV for measurement of peak area was found to be 0.64%. Repeatability of the method was checked by spotting 12 µl of standard solution seven times on TLC plate (n=7) and % CV for peak area was found to be 1.3%. Both the % CV, for measurement of peak area and sample applications (less than 1% and 3%, respectively), ensuring proper functioning of HPTLC system.

Accuracy of method was evaluated by calculating recovery of drug by standard addition method at 5 levels of the calibration curve (n=3). The percentage recovery was found to be 93.3 to 99.4% ensuring that the method is accurate.

The results indicate that the recovery of added sample was between 99.6 - 102.5 %. This clearly indicates that the method is accurate and precise.

The method is found to be specific for pioglitazone. The purity of the peak was determined by comparing the spectra at three different levels i.e. at peak start(S), peak apex (M) and peak end (E). Correlation between these three spectra indicated the purity of peak (correlation, r(S,M)=0.9999, r(M,E)=0.9993, fig. 2). The spectrum of extracted from tablet was also compared with spectrum of standard, which showed correlation 0.9993. It was observed that the excipients present in formulation did not interfere with the peak of pioglitazone.

Different validation parameters for the proposed HPTLC method for determining pioglitazone content are summarized in Table 1. This method was applied to determine the content of pioglitazone in two different market samples of single component pioglitazone tablets. The content and percentage of pioglitazone in two different market samples were found to be 30.03 mg, 100.1±2.9% and 29.82 mg, 99.4±1.5%, respectively (n=3). The results indicate that the proposed HPTLC method was found to be simple, specific, rapid, precise and accurate for estimation of pioglitazone in its formulations.

CONCLUSION:

The results indicate that the proposed method is simple, accurate, precise and specific, for estimation of pioglitazone in bulk and its formulations.

REFERENCES:

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4. Zhong, W.Z., Williams, M.G. Simultaneous quantitation of pioglitazone and its metabolites in human serum by liquid chromatography and solid phase extraction. J. Pharm. Biomed. Anal. 1996; 14: 465.

5. Zhong, W.Z., Lakings, D.B. Determination of pioglitazone in dog serum using solid- phase extraction and high-performance liquid chromatography with ultraviolet (229 nm) detection. J. Chromatogr. 1989; 30; 490(2): 377.

6. Sripalakit, P., Neamhom, P., Saraphanchotiwitthaya, A. High-performance liquid chromatographic method for the determination of pioglitazone in human plasma using ultraviolet detection and its application to a pharmacokinetic study. J. Chromatogr. B: Analytical Technologies 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 two active metabolites in human plasma by LC-MS/MS. J. Pharm. Biomed. Anal. 2003; 33 (1): 101-108.

Received on 24.03.2009 Modified on 15.04.2009

Asian J. Research Chem. 2(2): April.-June, 2009 page 207-209