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

Abstract

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.