Three methods are described for quantitative estimation of etizolam in bulk and in tablet dosage form by UV spectroscopy, HPTLC and RP-HPLC. In the first method, etizolam was estimated by measuring absorbance at λmax of 249 nm in 0.01 N HCl. In the second method, etizolam was estimated by HPTLC method using precoated plate of silica gel 60F254 and Toluene: Chloroform: Methanol (4.8:4.0:1.2 v/v/v) as mobile phase. The linear regression analysis data was used for the regression line in the range of 400–1600 ng/spot. In the third method, etizolam was estimated by simple and specific RP-HPLC method using the Kromasil column [C18 (5µm, 15 cm×4.6 mm, i.d.)] at ambient temperature using a mobile phase consisting of water: acetonitrile (50:50% v/v) and flow rate was 1.0 ml/min. Quantitation was achieved with UV detection at 241 nm based on peak area with linear calibration curves at concentration ranges 4–32 µg/ml etizolam. The specificity of HPLC method was confirmed by performing forced degradation study under thermal, humidity, acidic, alkaline, oxidative and photolytic conditions. The drug was found to degrade under acidic & oxidative conditions. The developed RP-HPLC method adequately separated the drug from the degradation products proving the specificity of method. The results of RP-HPLC method compared favourably with Japanese pharmacopoeia method. All three methods have been successfully applied to tablet formulation. All three methods were validated in terms of precision, recovery, limit of detection, limit of quantitation and robustness. The analysis of variance (ANOVA) and Student’s t-test were applied to correlate the results of etizolam determination in tablet dosage form by means of UV, HPTLC and RP-HPLC methods.
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