Simultaneous Determination of Sulphadiazine Sodium and Trimethoprim in Medicated Fish Feed, Fish Tissues and in Their Veterinary Pharmaceutical Formulation by Thin-Layer Chromatography-Densitometry

A specific, precise, and accurate thin-layer chromatographic method for the simultaneous estimation of sulphadiazine sodium (SDZ) and trimethoprim (TMP) in medicated fish feed and in fish tissues was developed and validated. This method is based on simple liquid extraction technique and employing thin-layer chromatography (TLC) as a cleanup step. In order to optimize the extraction procedure from fish tissues, several mobile phase systems and extracting solvents were tried. The method employed TLC aluminum plates precoated with silica gel 60 F-254 as the stationary phase and chloroform-toluene-ethanol-glacial acetic acid (4.5:4.5:1.0: 1.0 by volume) mixture as the developing solvent. This system was found to give compact and dense spots for both sulphadiazine sodium (R-F value of 0.48) and trimethoprim (R-F value of 0.16) without interference from either medicated fish feed or fish tissues coextractives. Densitometric analysis of both drugs was carried out in the reflectance-absorbance mode at 270 nm for SDZ and 225 nm for TMP to maximize sensitivity for each drug. The linearity of the proposed method was established over the ranges 0.1-2.0 and 0.1-1.0 mu g/band for sulphadiazine sodium and trimethoprim, respectively. The method was validated for linearity, specificity, precision, and accuracy. Statistical analysis proves that the method is repeatable and selective for the estimation of both drugs in various matrices. The proposed method was successfully applied for the determination of SDZ and TMP either in bulk pure powder or in their veterinary pharmaceutical formulation.