Validation of Commercial Formulation of Difenoconazole Using HPLC Equipped with Dad

Abstract

Effective, selective, precise, and accurate liquid chromatographic analytical methods for the determination of difenoconazole fungicide in the formulation have been optimized and validated

to meet the accreditation requirements, the performance characteristics of the analytical method were validated which is also one of the basic requirements of ISO Standard 17025:2017, for the rapid determination of difenoconazole by HPLC-DAD. The used method involves the extraction of the substances by sonication of the sample with acetonitrile, followed by dilution in acetonitrile, and direct injection on a liquid chromatography system. For the analysis LC system from Agilent Technologies 1290 Infinity II was used. Good separation was achieved on a Luna C18 column (3µm, 100°A, 3x150mm) with a guard column (C18 4x2.0mm I.D), an isocratic mobile elution consisting of acetonitrile and water acidified 0.075% with formic acid (85:15, v/v), at a flow rate of 0.7 ml/minute and UV detection at 220 nm. The column temperature was 25⁰C, injected volume was 1 μL. The analysis duration was 10 minutes (the retention time of difenoconazole and 4 – Hydroxybenzoic acid – methyl ester was 1.846 and 1.135 minutes, respectively. The linearity within the concentration range of 750-125 µg mL^-1, with the internal standard at a concentration of 250 µg mL^-1 with an average correlation coefficient (R²) of 0.9998. We have considered precision, repeatability, and selectivity in the validation.