Stability-indicating Methods for the Determination of Closantel Sodium by HPLC, Densitometry, Spectrofluorimetry and pH Induced ∆A

Abstract

Four stability-indicating methods were developed for the determination of closantel-Na in the presence of its alkaline degradation products. The first one was HPLC method in which efficient separation was achieved on a C18 analytical column with isocratic elution and a mobile phase composed of two mixtures A and B in a ratio of (30:70, v/v). Mobile phase A of ammonium acetate buffer- acetonitrile- water (100:30:870, v/v/v) and mobile phase B of the same composition in a ratio (100:870:30, v/v/v). Bromohexine-HCl was used as an internal standard and linearity was obtained in the range of 0.5-10.0 μg/mL with mean accuracy of 99.63% ± 0.49. The second method was a densitometric evaluation of thin-layer chromatograms of the drug using a mobile phase of ethyl acetate-methanol-ammonia (33%) (17:2:1, v/v/v) and scanned at 245 nm. Closantel-Na was separated from its three degradates with Rf 0.40, 0.05, 0.14 and 0.93, respectively; and determined in the range of 0.5-10.0 μg/spot with mean accuracy of 100.11%±0.27. The third method was based on measuring the native fluorescence intensity of closantel-Na in ethanolic H2SO4 solution at 477 nm (λex 337 nm) in the range of 0.5-5.0 μg/mL with mean accuracy of 99.84%±0.52. The forth one was pH-induced ΔA spectrophotometry based on measuring the ΔA of closantel-Na between ethanol and ethanolic HCl solution at 373.5 and 286.5 nm in the range of 6.0-100.0 μg/mL with mean accuracy of 99.43%±0.61 and 99.73%±0.58; respectively. The four methods provided selective recovery of the intact drug (99.24%±0.61, 100.21%±1.33, 100.54%±1.06 and 99.67%±0.96 and 100.50%±1.14; respectively.) in the presence of up to 90% its alkaline degradates by HPLC method and densitometric methods, 30% by the spectrofluorimetric method and 40% at λ max 373.5 nm and 15% at 286.5 nm, respectively; by the pH induced ΔA method. Successful application of the four methods for analyzing closantel-Na in Closa-Atak injectable solution was obtained and results were statistically analyzed and found to be in accordance with those given by a reported method.