Development of New Liquid Chromatographic Method for Mitotane and Its Metabolites Determination in Human Plasma Employing Design of Experiments Methodology

Abstract

A simple and reliable new HPLC method with UV detection has been developed and validated for simultaneous determination of mitotane and its two metabolites DDA and DDE. Method development was carried out utilizing systematical approach of the design of experiments (DoE) methodology. For estimation of factors influence on selected chromatographic responses and definition of the optimal chromatographic conditions, Box–Behnken experimental design was applied. The defined optimal separation conditions were: column Restek Ultra Aqua C₁₈ with pre-column Restek Ultra Aqua C₁₈ operating at temperature 35°C; mixture of acetonitrile and 0.5% formic acid as mobile phase with 1.2 mL min⁻¹ flow rate and detection at 230 nm. As sample preparation method, liquid–liquid extraction was chosen. Method was fully validated and LOQ and LOD were experimentally determined. Finally, method was successfully applied for determination of mitotane and its metabolites in plasma samples of patients with adrenocortical carcinoma.

Keywords:

• experimental design
• liquid chromatography
• metabolites DDA and DDE
• mitotane
• validation