Development and validation of a stability-indicating HPTLC method ...

Dinesh Kumar Jain et al., Int J Pharm Biomed Sci 2011, 2(2), 55-60Table 1System suitability parameter for HPLC methodParameters LOR PCMCalibration range (µg/mL) 2-40 8-150Theoretical plate number 4009 2311HETP a 0.0073 0.004Tailing factor 1.52 1.86Capacity factor (k’) 0 1.78Resolution - 6.58a HETP = Height equivalent to theoretical plate, cmTable 2Summary of validation dataS. No. Validation parameters Observation of HPTLC methodLORPCM1 Linearity range300-550 100-500(ng/band)2 Slope 10.48 4.663 Intercept 1226.7 -137.044 Wavelength (nm) 249 3705 Correlation coefficient 0.993 0.9966 Accuracy (n=5) 100.39±1.42 100.56±1.27 Intraday Precision (n=3) 1971.7±24.85 1356.6±23.378 Interday Precision (n=3) 5376.6±88.37 1361.6±35.549 Repeatability (n=6) 3814.8±66.61 313.88±6.3410 Recovery (n=3) 98.91±1.39 98.84±0.5711 LOD (ng/band) 0.62±0.02 0.92±0.0312 LOQ (ng/band) 1.26±0.01 2.17±0.0213 Robustness Robust Robust14 Specificity Specific Specific2.3.5 Limit of detection (LOD) and Limit of quantitation(LOQ)The LOD with signal-to-noise (S/N) ratio of 3:1 and theLOQ with S/N ratio of 10:1 were calculated for LOR andPCM using the following equations LOD = 3.3 σ/S, LOQ =10 σ/S where σ is the standard deviation of the response andS is the standard deviation of the y-intercept of the regressionline for HPLC analysis. For HPTLC analysis the limit ofdetection (LOD) and the limit of quantification (LOQ) wasdetermined by diluting known concentration of standardstock solution until the average responses wereapproximately 3 or 10 times the responses of the blank.2.3.6 RecoveryTo check the recovery of the drug at different levels informulations, samples were spiked with 50, 100, and 150 %of LOR and PCM standards and the mixtures were analyzedin triplicate by the proposed method.2.4 Stability of Sample SolutionsSolutions at two different concentrations (300 and350ng/band for LOR and 200 and 300ng/band for PCM)were prepared from stock solution and stored at roomtemperature for 24, 4.0, 2.0 1.0, 0.5 and 0.2h. They were thenchromatographed on the same plate. The chromatogramswere evaluated for additional spots if any.2.5 Assay of the marketed formulationTwenty tablets were weighed and crushed. An amount ofpowder equivalent to 100 mg PCM was transferred to a 100mL volumetric flask and extracted with methanol for 30 minby shaking mechanically and volume was adjusted to markwith the same solvent. 1mL of this solution was diluted to 10mL with methanol. An appropriate volume of 5 μL wasassayed by the chromatographic procedure described above.No interferences were observed from the excipientscommonly present in the tablets.2.6 Forced degradation studiesA stock solution containing 10 mg for each (LOR andPCM) in 10 mL methanol was prepared. This solution wasused for forced degradation to provide an indication of thestability-indicating property and specificity of the method.The average peak area of sex replicates of LOR and PCM(1000 ng/band) was used for analysis.2.6.1 Acid degradationMethanolic solution of each drug (10 mg) was separatelydissolved in 10 mL of 1M HCl and these solutions were keptfor 8 h at room temperature in dark in order to exclude thepossible degradative effect of light. The solutions (1 mL)were taken and neutralized and then diluted up to 10 mL withmethanol. The resultant solutions were applied on TLC platein triplicate (10 μL each, i.e. 1000 ng/band). The plate waschromatographed as described above.2.6.2 Base degradationMethanolic solution of each drug (10 mg) was separatelydissolved in 10 mL of 1 M NaOH solution. These solutionswere kept for 8 h at room temperature in dark in order toexclude the possible degradative effect of light. The solutions(1 mL) were taken and neutralized and then diluted up to 10mL with methanol. The resultant solutions were applied onTLC plate in triplicate (10 μL each, i.e. 1000 ng/band). Theplate was chromatographed as described above.2.6.3 Oxidative degradationEach drug (10 mg) was dissolved in 10 mL of methanolicsolution of hydrogen peroxide (10% v/v) and kept for 8 h atroom temperature in the dark, to exclude the possibledegradative effect of light. The solution (1 mL) was thendiluted to 10 mL with methanol and treated as described foracid and base-induced degradation.57©2011 PharmaInterScience Publishers. All rights reserved. www.pharmainterscience.com