HPTLC Method for Simultaneous Determination of Pioglitazone HCl and Telmisartan in Tablet Dosage Form
P Kalaiselvi*, R Vijay Amirtharaj, T Venkatachalam and N Senthil Kumar
JKKMMRF College of Pharmacy, Komarapalayam -638183. Tamil Nadu, India.
*Corresponding Author E-mail: kalaimpa@yahoo.co.in
ABSTRACT:
A simple, precise, accurate and rapid HPTLC method has been developed, and validated for the determination of Pioglitazone HCl and Telmisartan simultaneously, in combined dosage form (15mg and 20mg). Pioglitazone HCl and Telmisartan was chromatographed on silica Gel 60F254 TLC plate using Toluene: ethyl acetate: methanol (7:2:1v/v) was used as the mobile phase. The Rf value of Pioglitazone HCl and Telmisartan was 0.56±0.04 and 0.23±0.02 and scanned at 254nm using camag TLC scanner 3. The applicability of the method for simultaneous determinations of Pioglitazone HCl and Telmisartan was verified by the determination of these compounds in marketed tablets. Results of the analysis were validated statistically, and by recovery studies (99.47-101.36% and 99.40 -102.37%). The recovery and RSD values were with in limits given in ICH guidelines, method developments indicates that the suitability of proposed for the routine determination of these compounds in tablets. The validation parameters; linearity the range 30-150 ng/spot and 40-200ng/spot. (r=0.9995 and r=0.9996) sensitivity. The limit of detection was found to be 15ng/spot and 20ng/spot. The limit of quantification was found to be 45ng/spot and 60ng/spot respectively. The accuracy ((99.88% and 99.33%) and reproducibility were found to satisfactory. The proposed method can be successfully used to determine the drug contents on marketed formulation.
KEYWORDS: HPTLC, Pioglitazone HCl, Telmisartan, Methanol, Validation
INTRODUCTION:
Pioglitazone hydrochloride chemically, [5-(CH-C2-C5-ethyl 2-pyridinyl) ethoxy) phenyl) methyl)-2, 4] thiozolidine Dione monohydro chloride1. It is used as an anti diabetic drug. Telmisartan is chemically 41[Cl4 –Dimethyl-2-propyl [2, 6-bi-1H-benzimideazol]-1-yl] methyl (1, 1-biphenyl)-2-Carboxylic acid1. It is used as treatment of hypertension. Literature survey revealed that there is no analytical method reported for estimation of Pioglitazone hydrochloride and telmisarton in combination; reported methods2-11 were discussing the analysis either individually or with other combination by spectrometry and HPLC in biological fluids and pharmaceutical dosage forms. Pioglitazone hydrochloride and telmisartaon has recently been introduced into the market. Our study made an attempt to develop, simple, accurate and reproducible HPTLC method for routine analysis of these drugs simultaneously in tablet dosage form.
MATERIALS AND METHODS:
Pioglitazone hydrochloride and Telmisartan standard and formulations were procured as gift sample from the Madras pharmaceuticals, Madras-96, silica gel 6 0f 254 on aluminum sheets (Merck Mumbai) layer thickness 0.2mm were used as a stationary phase. All chemicals and reagents used were of analytical grade. The tablets containing Pioglitazone hydrochloride (15gm) and Telmisartan (20 gm) was procured from Madras pharmaceuticals the a Camag HPTLC system comprising of Camag Linnomate IV semiautomatic sample applicator, Hamilton syringe (100 let), Camag TLC Scanner 3, Camag win CATS4 software, Camag Twin-through chamber (20x10 cm) and ultrasonicator were used during study. All the chemical and solvents were of analytical reagent grade. Toluene, Ethyl acetate, methanol.
Preparation of standard solution of Pioglitazone HCl and Telmisartan:
Pioglitazone HCl (15mg) and Telmisartan (20mg) were accurately weighed and dissolved in methanol and make up with 100ml in volumetric flask. A standard stock solution of Pioglitazone HCl (150 µg/ml) and Telmisartan (200µg/ml) were prepared.
Preparation sample solution:
Twenty tablets were weighed and finely powdered. The powder equivalent to 15mg Pioglitazone HCl and 20mg Telmisartan was weighed accurately and mixed with 5 ml methanol and sonicated for 10 minutes. The solution was filtered through what man no.14 filter paper and washed. The filtrate and washings were combined in 100ml volumetric flask and made up to the mark with methanol.
HPTLC method and chromatographic condition:
The chromatographic estimations were performed using following conditions; stationary phase, percolated silica gel 60F254 aluminum sheets (20x20 cm) rewashed with methanol, and dry in air] mobile phase Toluene : ethyl acetate : methanol (7:2:1%v/v/); chamber saturation time, 30 minutes, temperature, 25±2°C; migration distance, 80mm wavelength of detection, 254nm slit dimensions, 6x0.45mmscanning speed 5mm/second. A deuterium lamp is the source of radiation. 6µl of standard solutions of Pioglitazone HCl and temisartan were spotted and developed at constant temperature and wavelength was selected by scanning standard solution of both drugs over 200nm to 400nm wavelength. Pioglitazone HCl show maximum 269 nm and Telmisartan show maximum absorbance at 299 nm. Both components show reasonably good response at 254 nm. Therefore photometric measurements were performed at 254 nm in reflectance mode with Camag TLC scanner 3 using wincats software.
Calibration curve:
Standard Pioglitazone HCl and Telmisartan 2, 4, 6, 8, 10, µl was spotted on percolated separate TLC plate, using semiautomatic spotter under nitrogen steam. The TLC plates were developed and photometrically analyzed as described under chromatographic separation. The calibration curves were prepared by plotting peak area versus concentration (ng/spot) corresponding to each spot.
Quantification of dosage forms:
Sample solution 6μl [90µg/ml and 120µg/ml] was applied on TLC plate, developed and scanned as described in chromatographic separation. The amount of Pioglitazone HCl and Telmisartan present in sample was determined by fitting area values for peak corresponding to Pioglitazone HCl and Telmisartan into the equation of line representing calibration curve for Pioglitazone HCl and Telmisartan.
RESULTS AND DISCUSSION:
In present work HPTLC method was developed for estimation of Pioglitazone HCl and Telmisartan pure and for dosage forms. HPTLC method is cost effective and less time consuming. Pioglitazone HCl and Telmisartan is soluble in methanol, therefore methanol was selected as solvent. The formulation was dissolved in methanol with sonication for 10min to assure complete release of drug from the formulation.
Method optimization:
For optimization of method, different mobile phase compositions were employed to achieve good separation. The method development was initiated by using a mobile phase of Chloroform: methanol, methanol: water: conc. HCl; various proportions they showed poor separation. In the above conditions, elution was very poor and broad. Introduction of acetic acid in above mobile phase gave sharp peaks but poor separation and band broadening was observed. Early elution with little separation was observed with the mobile phase consisting of Toluene: ethyl acetate: methanol (2:8:0.5) and Toluene: ethylacetate: methanol (7:1.5:1) gave reasonable Rf but not sharp band. There fore needed further optimization on the other proportion finally mobile phase containing Toluene : ethylacetate : methanol (7:2:1) could resolve Pioglitazone HCl and Telmisartan spot with better peak and sharp. The Rf value of this mobile phase in Pioglitazone HCl and Telmisartan was 0.56±0.04 and 0.23±0.02 and also resolution of dosage forms from matrix. Even saturation of TLC chamber with mobile phase for 30 minute assured better reproducibility and better resolution. Pioglitazone HCl and Telmisartan shows significant UV absorbance at wavelength 254nm.
The method was validated12 in terms of linearity, interday and intraday 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 HCl and Telmisartan was obtained by plotting the mean peak area of Pioglitazone HCl and Telmisartan against the concentration over the range 30-150 µl/ml and 40-200µl/ml. A correlation coefficient was found to be 0.9995 and 0.9996 and RSD ranging from 0.31 and 0.33. The average linear regression equation was represented as Y=3.38X-291 and Y=2.5425X+1893, where X- concentration of Pioglitazone HCl and Telmisartan, Y=peak area. The limit of detection was found to be 15ng/spot and 20ng/spot. The limit of quantification was found to be 45ng/spot and 60ng/spot, respectively. Intraday and interday variation range for Pioglitazone HCl and Telmisarton was found to be, 1.01 and 0.47, 0.805 and 0.29. Precision of the instrument was checked by repeated scanning of same spot 200ng/spot for six times without changing position of the plate and % CV for measurement of peak area was found tobe0.70 and0 .36, respectively. Repeatability of the method was checked by spotting 10µl/ml sample solution six times on TLC plates (n=6) and % CV for peak area was found to 0.30 and 0.60, respectively, ensuring proper functioning of HPTLC system. Accuracy of method was evaluated by calculating recovery of drug by standard addition method at 2 levels of the calibration curve (n=6). The % recovery was found to be 99.47 -101.36 and 99 .40 -102.37 Pioglitazone HCl and Telmisartan ensuring that this method is accurate. The method is found to be specific for Pioglitazone HCl and Telmisartan. Different validation parameters for the proposed HPTLC method for determination the Pioglitazone HCl and telmisartan content was summarized in Table 1.
|
S. No |
Parameters |
Pioglitazone HCl |
Telmisartan |
|
1 2. 3.
4. 5 6.
7. 8. 9. 10. 11. |
Linearity range (ng/spot) Correlation Coefficient(r2) Precision a) Intra day b) Inter day Slope(m) Intercept(c) Repeatability of sample application (%RSD) (n=6) Repeatability of Peak area(n=6) LOD LOQ % Recovery Specificity |
30-150ng/spot 0.9995
1.01 0.805 3380 2910 0.309
0.70 15ng/spot 45ng/spot 99.47-101.36 specific |
40-200ng/spot 0.9996
0.47 0.29 2540 18930 0.33
0.36 20ng/spot 60ng/spot 99.40-102.37 specific |
Table: 1 Summary of validation parameters
This method was applied to determine the content of Pioglitazone HCl and Telmisartan label claim 15mg and 20mg were found to be 14.98mg and 19.86mg; 99.88% and 99.33%, 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 HCl and Telmisartan and in its dosage forms.
CONCLUSION:
The results indicate that the proposed method is simple, accurate, precise and specific for estimation of Pioglitazone HCl and Telmisartan in bulk and its dosage forms.
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Received on 24.08.2009 Modified on 15.10.2009
Accepted on 20.11.2009 © AJRC All right reserved
Asian J. Research Chem. 3(1): Jan.-Mar. 2010; Page 60-62