Insights into the separation, enantioseparation and recognition mechanisms of amphetamineand methamphetamine isotopologues on achiral and polysaccharide-based chiral columns inhigh-performance liquid chromatography. First Baseline Separation of Isotopomers.

Overview

This study addresses the simultaneous chromatographic separation of enantiomers and isotopologues of amphetamine and methamphetamine derivatives using high-performance liquid chromatography on both achiral and polysaccharide-based chiral stationary phases. The work demonstrates baseline separation of partially deuterated isotopologues and enantioisotopologues, achieving a previously unreported resolution of isotopomers under conventional HPLC conditions.

Methods and approach

Separation experiments were conducted on achiral columns for isotopologue discrimination and on polysaccharide-based chiral columns for simultaneous enantiomeric and isotopic resolution. Systematic variation of mobile phase composition was employed, including acetonitrile and methanol-based systems with different aqueous components and additives. Parameters investigated included stationary phase chemistry, mobile phase polarity and pH, separation temperature, and structural positioning of deuterium atoms within the amphetamine scaffold. Molecular modeling was utilized to elucidate retention mechanisms and stereochemical interactions.

Results

Baseline separation of deuterated amphetamine and methamphetamine isotopologues was achieved on achiral columns, with isotope effects demonstrating pH-, solvent-, and temperature-dependent behavior. Mobile phase composition critically influenced retention discrimination, with acetonitrile-containing systems producing stronger isotope effects than methanol-based phases. The isotope effect exhibited switchable behavior between normal and inverse modes depending on mobile phase composition. Polar, hydrogen bonding-type interactions predominated in normal isotope effects within polar organic solvents, while apolar hydrophobic interactions mediated inverse isotope effects favored by increasing aqueous content in reversed-phase systems. Polysaccharide-based chiral columns enabled simultaneous separation of enantiomeric and isotopic species, with isotopomer elution order reversible on specific chiral stationary phases.

Implications

The demonstration of isotopomer separation under standard HPLC conditions extends analytical capabilities for compounds where kinetic isotope effects have biomedical significance. The mechanistic understanding of normal versus inverse isotope effects—governed by distinct noncovalent interaction types—provides a framework for rational mobile phase optimization in future isotopologue and enantioisotopologue separations. This work validates polysaccharide chiral selectors for complex isotopic and enantiomeric discrimination simultaneously. The results have applicability to both analytical-scale isotopomer characterization and preparative-scale enrichment of isotopically labeled compounds. The tunable nature of isotope effects through mobile phase engineering enables control of retention and selectivity independent of structural modification.