Experimental Design Approach for Development of HPLC Method for Simultaneous Analysis of Triamcinolone, Nystatin, and Gramicidin in Industrial Wastewater

Abstract

This study used an experimental design approach to optimize an HPLC method for the simultaneous determination of three pharmaceutical residues (triamcinolone, nystatin, and gramicidin) in industrial wastewater samples. The goal of using an experimental design approach was to maximize the method performance through separation enhancement and shortening the time of analysis and/or minimizing the environmental effects through the reduction in wastes and sample treatment. To achieve this goal, two steps were performed: a full factorial screening design for the three chromatographic variables, and optimization design using central composite design to select the optimum conditions that accomplished the highest resolution between adjacent peaks within a minimum run time of less than 5 min. The optimal chromatographic conditions derived from Minitab software using the desirability function were applied. Separation was carried out on a Zorbax C18 column (250 mm × 4.6, 5 μm) with gradient elution of a mobile phase composed of methanol and 0.25 M potassium dihydrogen phosphate buffer (pH 3.6) at different UV detections. For the validation of the developed HPLC method, ICH guidelines were followed, and the obtained results were found to be in compliance with the acceptance criteria. Linearity was over the concentration range of 1.00–25.00 μg/mL for triamcinilone and nystatin and 10.00–50.00 µg/mL for gramicidin. The proposed method was successfully applied to quantify the three studied pharmaceutical compounds in rinsing wastewater samples.