INTRODUCTION:

Rosiglitazone 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. Rosiglitazone 5-((4-(2-(methyl-2-pyridinylamino) ethoxy)phenyl) methyl)- 2,4-thiazolidinedione belongs to a different chemical class and has a different pharmacological action than the sulfonylureas, metformin, or the α- glucosidase inhibitors¹.

Rosiglitazone is not official in any of the pharmacopoeia. Literature survey reveals that one spectrophtometric method², four HPLC methods in human plasma^3-6, one HPLC method in tablet dosage form⁷and one HPTLC method in its dosage form⁸. The aim of this work was to develop and validate simple, specific, sensitive, accurate, precise, rapid and cost effective HPTLC method for the estimation of rosiglitazone in bulk and its formulation.

MATERIALS AND METHODS:

Materials:

Rosiglitazone working standard was procured as a gift sample from Torrent Reseach center, 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 rosiglitazone (4 mg) (formulation A- Rosicon, manufactured by Torrent Pharmaceuticlal industries Ltd., Ahmedabad, Formulation-B Result, manufactured by Sun Phrma Ltd., Baroda) were purchased from market. Toluene, ammonia (SD’S) and acetone (A.R., Finar) 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 rosiglitazone:

Rosiglitazone (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 rosiglitazone per ml of the solution (S1).

Fig. 1 Chromatogram of rosiglitazone from tablet Chromatogram of the sample showing resolution of rosiglitazone peak (800 ng/spot, Rf =0.55) from components of formulation matrix.

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

Preparation of sample solution:

Ten tablets were weighed and finely powdered. The powder equivalent to rosiglitazone (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:acetone:ammonia (4.5:5.5:0.1 v/v); chamber saturation time, 30 min; Temperature, 29±4^o; migration distance, 45 mm; wavelength of detection, 318 nm; slit dimensions, 3×0.45 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 : acetone : ammonia (4.5:5.5:0.1 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 318 nm in absorbance/reflectance mode with Camag TLC Scanner 3 with CATS4 software incorporating the track optimization option.

Calibration curve of standard rosiglitazone:

Standard rosiglitazone solution (4, 8, 12, 16, 20, 24, 28, 32 and 40 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 rosiglitazone in tablet formulation:

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

Fig. 2 Peak purity spectra of standard rosiglitazone Peak purity spectra of standard rosiglitazone (800 ng/spot) at peak start, peak apex and peak end.

RESULTS AND DISCUSSION:

In present work HPTLC method was developed for estimation of rosiglitazone pure powder and its pharmaceutical formulation. HPTLC method is cost effective and less time consuming. Rosiglitazone 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 proportions. In the above conditions elution was very broad for rosioglitazone. Introduction of ethyl acetate in the above mobile phase 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: ammonia (7:3:0.1) gave more sharp band but change the Rf by slight changing the proportion of methanol. Therefore need further optimization, in above mobile phase acetone was used in place of methanol gave better separation. Finally, the mobile phase consisting of the mixture of toluene: acetone: ammonia (4.5:5.5:0.1 v/v) could resolve rosiglitazone spot with better peak shape. Combination of toluene and acetone offered optimum migration (Rf= 0.55±0.03) and resolution of rosiglitazone from other components of formulation matrix. Even saturation of TLC chamber with mobile phase for 30 min assured better reproducibility and better resolution. Rosiglitazone shows significant UV absorbance at wavelength 318 nm. Hence this wavelength has been chosen for detection in the analysis of rosioglitazone.

Table: 1 Summary of validation parameters:

Sr. No.	Parameters	Results
1	Linearity range (ng/spot)	200-2000 ng/spot
2	Correlation co-efficient	0. 9900
3	Precision
	Intra-day % CV (n = 3)	0.6-3.6
	Inter-day % CV (n =3)	0.6-3.0
4	Repeatability of sample application (n = 7)	1.25
5	Repeatability of peak area ( n = 7)	0.29
6	% Recovery	97.1 – 103.3
7	Limit of detection	30 ng/spot
8	Limit of quantification	100 ng/spot
9	Specificity	Specific

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 rosiglitazone was obtained by plotting the mean peak area of rosiglitazone against the concentration over the range of 200 - 2000 ng/spot. A correlation coefficient was found to be 0.9900 and RSD was ranging from 0.6- 3.6. The average linear regression equation was represented as Y=4.7558X+1760.8, where X=concentration of rosiglitazone and Y=peak area. The limit of detection and limit of quantification for rosiglitazone were found to be 30 ng/spot and 100 ng/spot, respectively.

Inter-day and Intra-day variation range for rosiglitazone was found to be 0.6 - 3.0 and 0.6 - 3.6 respectively. Precision of the instrument was checked by repeated scanning of the same spot (1000 ng/spot) of seven times without changing position of the plate and % CV for measurement of peak area was found to be 0.29%.

Repeatability of the method was checked by spotting 16 µl of standard solution seven times on TLC plate (n=7) and % CV for peak area was found to be 1.25%. 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 3 levels of the calibration curve (n=3). The percentage recovery was found to be 97.1 to 103.3% ensuring that the method is accurate.

The method is found to be specific for rosiglitazone. 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)=0.9997, fig. 2). The spectrum of extracted from tablet was also compared with spectrum of standard, which showed (correlation, r(S,M)=0.9999, r(M,E)=0.9954). It was observed that the excipients present in formulation did not interfere with the peak of rosiglitazone. Different validation parameters for the proposed HPTLC method for determining rosiglitazone content are summarized in Table 1. This method was applied to determine the content of rosiglitazone in two different market samples of single component rosiglitazone tablets. The content and percentage of rosiglitazone in two different market samples were found to be 3.93 mg, 98.3±0.28% and 4.06 mg, 101.6±0.57%, respectively (n=3). The results indicate that the proposed HPTLC method was found to be simple, specific, rapid, precise and accurate for estimation of rosiglitazone in its formulations.

CONCLUSION:

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

REFERENCES:

1. Budavari S, editor. The Merck Index. 13^th ed. Whitehouse Station (NJ): Merck and Co Inc; 2001. 1484.

2. Gomes, P., Sippel, J., Jablonski, A., Steppe, M. First-derivative spectrophotometry in the analysis of rosiglitazone in coated tablets. Journal of Pharmaceutical and Biomedical Analysis, 2004, 36 (4), 909-913.

3. Mamidi, R.N.V.S., Benjamin, B., Ramesh, M., Srinivas, N.R. HPLC method for the determination of rosiglitazone in human plasma and its application in a clinical pharmacokinetic study. Biomedical Chromatography, 2003, 17 (6), 417-420.

4. Kolte, B.L., Raut, B.B., Deo, A.A., Bagool, M.A., Shinde, D.B. Liquid chromatographic method for the determination of rosiglitazone in human plasma. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 2003, 788 (1), 37-44.

5. Muxlow, A.M., Fowles, S., Russell, P. Automated high-performance liquid chromatography method for the determination of rosiglitazone in human plasma. Journal of Chromatography B: Biomedical Sciences and Applications, 2001,752 (1), 77-84.

6. Kim, K.A., Park, J.Y. Simple and extractionless HPLC determination of rosiglitazone in human plasma and application to pharmacokinetic to human plasma. Biomedical Chromatography, 2004, 18 (8), 613-615.

7. Radhakrishna, T., Satyanarayana, J., Satyanarayana, A. LC determination of rosiglitazone in bulk and pharmaceutical formulation. Journal of Pharmaceutical and Biomedical Analysis, 2002, 29 (5), 873-880.

8. Sane, R.T., Francis, M., Moghe, A., Khedkhar, S., Anerao, A. High-performance thin-layer chromatographic determination of rosiglitazone in its dosage form. Journal of Planar Chromatography - Modern TLC, 2002, 15 (3), 192-195.

Received on 09.06.2009 Modified on 11.08.2009

Asian J. Research Chem. 3(1): Jan.-Mar. 2010; Page 26-28