Validation of Thin-Layer Chromatography-Bioautographic Method for Determination of Streptomycin

Background: A simple bio-assay for determination of streptomycin hyphenated with planar chromatography techniques was developed. Objective: This study aims to validate the method for identification and determination of streptomycin in injection preparations with TLC-bioautography. Methods: Thin Layer Chromatography (TLC) was performed on the silica Gel GF-254 using KH₂PO₄ solution as mobile solvent. The visualization was performed by spraying 2% resorcinol. Direct bi autography was developed using Escherichia coli ATCC 25922 as a bacterial test, grown on the nutrient agar medium at 37^oC for 24 hours. The method was validated corresponding to linearity, limit of detection (LOD), intra day precision, and accuracy parameters. The accuracy was measured using streptomycin injection as a sample. Results: The Results showed that the KH₂PO₄ solution at 7.5% concentration was found to be the optimized solvent with Rf value of 0.5. The linear equation was y = 10.176x + 4.046 at 150 - 350 µg/mL concentration range with the linearity coefficient, Limit of Detection, accuracy, and variation coefficient were 0.9907; 40 ppm; 96.37 + 2.22% (with an RSD value of 2.31%); and 1.63 respectively. Conclusion: The prospective TLC-bioautographic method was applied for the identification and determination of streptomycin in a preparation using a single eluent KH₂PO₄. The eluent system optimization remains necessary for the identification and determination of the mixture of streptomycin with other antibiotics, such as aminoglycoside groups.