Development and validation of a method for quantitative determination of the genotoxic impurity diethyl sulfate in pitolisant hydrochloride via high-performance liquid chromatography

Abstract

A simple and highly sensitive method was devised to detect and measure minute concentrations of diethyl sulfate (DES), a potential genotoxic impurity found in the active pharmaceutical ingredients of pitolisant hydrochloride. High-performance liquid chromatography (HPLC) was employed to accurately quantify this impurity, involving pre-column derivatization with sodium phenoxide. Chromatographic separation was achieved using a Shim-pack C18 column (250 × 4.6 mm ID × 5 μ), with a mobile phase composed of 0.01 M sodium dihydrogen orthophosphate in water (mobile phase A) and acetonitrile (mobile phase B) in a gradient elution mode at a flow rate of 1.5 mL min⁻¹ and the column temperature maintained at 25 °C. Detection was performed at 218 nm with an injection volume of 30 μL. Validation was conducted in accordance with standard International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines, encompassing parameters such as system suitability, specificity, the limit of detection (LOD), the limit of quantification (LOQ), linearity, and accuracy. The method demonstrated an LOD and LOQ of 4 ppm and 12 ppm, respectively. This developed HPLC methodology proved to be well-suited for quantifying trace levels of the potential genotoxic impurity diethyl sulfate (DES) in pitolisant hydrochloride, a by-product originating from the synthesis process.