Chiral HPLC method optimization for enantioseparation of 2-methylmethcathinone

Abstract

Background: 2-Methylmethcathinone (2‑MMC) is a chiral new psychoactive substance whose enantiomers may differ in pharmacological and toxicological behavior. Reliable enantioselective analysis is therefore required for forensic and environmental applications. Recent work shows that polysaccharide-based chiral stationary phases (CSPs) are broadly effective for cathinones [1]. Objective: This study aims to develop and optimize a rapid, robust high-performance liquid chromatography with ultraviolet detection (HPLC‑UV) method achieving baseline separation [resolution (Rs) ≥ 1.5] of 2‑MMC enantiomers. Methods: A two‑stage workflow was used. First, a screening compared a Lux AMP 3 µm (150 × 4.6 mm) under ammonium bicarbonate (pH 11) with methanol or acetonitrile (isocratic/gradient) against a Lux Amylose‑1 3 µm (150 × 4.6 mm) operated in normal‑phase (n‑hexane/isopropanol). Ultraviolet detection was set at 254 nm. Performance criteria included Rs and run time. Results: The Lux AMP configuration yielded limited enantioresolution across tested conditions (maximum Rs ≈ 1.11). In contrast, Lux Amylose‑1, with n‑hexane/isopropanol as mobile phase, produced baseline separation of 2‑MMC enantiomers (Rs > 1.5) with short analysis times and consistent retention, providing suitable peak shape and repeatability. These outcomes align with literature showing high success rates of amylose/cellulose CSPs for cathinones under normal‑phase and polar‑organic modes [2,3]. Conclusions: The screening‑to‑optimization strategy delivered a fast enantioselective HPLC‑UV method for 2‑MMC. Beyond analytical separation, the method provides a robust platform to develop and adapt enantioselective procedures for biological samples (e.g., oral fluid, blood, urine), enabling enantiomer‑resolved quantification in clinical and forensic toxicology.