Simultaneous Determination and Method Validation of Fluconazole and its impurities by High Performance Thin Layer Chromatography Using Reflectance Scanning Densitometry

P. Sreeman Narayana, A. Shiva Reddy, B. Ramesh, P. Sita Devi

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Densitometry, Fluconazole, Impurities, HPTLC, Method validation


A selective method based on HPTLC using reflectance scanning densitometry was developed and validated for the simultaneous separation and quantification of Fluconazole in formulations and its structurally related impurities. The chromatographic separation was accomplished on TLC aluminum plates precoated with silica gel 60 F254 as the stationary phase using a saturated mixture of butanol: water: acetic acid (8:2:1 v/v). For visualisation, the plate was dipped in to a modified anisaldehyde reagent and heated at 120°C for 30 minutes in a drying oven. Densitometric quantification was performed at 254 nm by reflectance scanning. Fluconazole appeared as a brown spot (Rf 0.67±0.02) and resolved well from the two impurities. The standard fluconazole curve is linear (r =>0.9995) over a concentration range of 100-800ng/spot. Recovery from tablet formulation was statistically equal to 100% .The limits of detection and quantification were 91.39 and 304.66 ng/spot respectively. The precision of the method with respect to concentration is acceptable with a relative standard deviation of 0.73%. The proposed method is specific for fluconazole in the presence of its structurally related impurities and proved to be a valuable complimentary method for impurity profiling and quality control.