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

## TL;DR

This paper presents a sensitive HPLC method to detect and measure diethyl sulfate, a harmful impurity in pitolisant hydrochloride.

## Contribution

A novel HPLC method with pre-column derivatization for quantifying genotoxic impurity diethyl sulfate in pitolisant hydrochloride.

## Key findings

- The method achieved a limit of detection (LOD) of 4 ppm and a limit of quantification (LOQ) of 12 ppm.
- The HPLC method was validated according to ICH guidelines and proved suitable for trace-level quantification of diethyl sulfate.

## 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.

Diethyl sulfate, a carcinogenic by-product in pitolisant hydrochloride synthesis, is converted to ethoxybenzene using sodium phenoxide, enabling its quantification by HPLC-UV to help minimize risk in the drug substance.