Baseline Separations of Isotopologoues of Amphetamine Derivatives Using HPLC

A recent study published in the Journal of Chromatography A performed a baseline separation of isotopologoues of some amphetamine (AMP) derivatives on achiral and polysaccharide-based chiral columns.

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Isotopically labelled compounds have become increasingly notable in the pharmaceutical industry in the last decade. Particularly, more research is being done to examine the kinetic isotope effect in biological systems, specifically in humans. The kinetic isotope effect (KIE) is a phenomenon associated with isotopically substituted molecules exhibiting different reaction rates. Effects such as these are used to aid in the understanding of reaction kinetics, mechanisms, and solvent effects (2). Undeuterated and deuterated biologically active compounds may have different pharmacokinetic, toxicokinetic, and most likely even pharmacodynamic properties. Such differences led to the approval of some deuterated chemical species as drugs for clinical use by regulatory bodies, such as deuruxolitinib and lomedeucitinib, since 2017 (3). Hydrogen/deuterium (H/D) isotope effects are not an unusual side effect in chromatography, with such phenomena being observed in gas- and liquid-phase separations. While there are numerous reports on this topic, there is little understanding of the mechanisms and underlying noncovalent interactions at play.

In a prior study, the scientists, from Università Politecnica delle Marche in Ancona, Italy, observed that the isotope effect was tunable and dependent on mobile phase composition in high-performance liquid chromatography (HPLC) for several amphetamine (AMP) derivatives. Under the studied conditions, stronger isotope effects were found in acetonitrile-containing mobile phases compared to methanol-containing ones with both chiral and achiral columns and both, “normal” and “inverse” isotope effects were observed. “Normal” effects were favored in polar organic solvents, while “inverse” isotope effects were found while increasing the content of the aqueous component in the reversed-phase mobile phase.

For this study, the scientists reported baseline separation of isotopologoues of some amphetamine (AMP) derivatives on achiral and polysaccharide-based chiral columns, as well as some correlations between the degree of separation of enantiomers and isotopologues on (the same) polysaccharide-based chiral column(s). In this frame, the impact of the number of deuterium atoms part of AMP isotopologues, the structure of isotopomers, the chemical structure of the achiral and chiral stationary phases used in this study, and the use of methanol- vs. acetonitrile-containing mobile phases on the isotope effects were reported and examined. Quantitative correlations between the isotope effect and the enantioselectivity of the chiral columns used were also shortly discussed.

Altogether, strong isotope effects were found for partially deuterated AMP derivatives under some experimental conditions, enabling baseline separation on achiral and polysaccharide-based chiral columns using HPLC. The nature (positive or negative) of the isotope effect and its extent strongly depends on the nature of selector, medium and structure of the studied compounds. On chiral columns some correlations were observed between the strength of the isotope effect and selectivity of enantioseparation.

References

(1) Sprega, G.; Kobidze, G.; Lo Faro, A. F.; Sechi, B.; et al. Separation of Isotopologues of Amphetamine with Various Degree of Deuteration on Achiral and Polysaccharide-Based Chiral Columns in High-Performance Liquid Chromatography. J. Chromatogr. A 2024, 1730, 465062. DOI: 10.1016/j.chroma.2024.465062

(2) Kinetic Isotope Effects. LibreTexts Chemistry 2024. https://chem.libretexts.org/Bookshelves/Physical_and_Theoretical_Chemistry_Textbook_Maps/Supplemental_Modules_(Physical_and_Theoretical_Chemistry)/Quantum_Mechanics/06._One_Dimensional_Harmonic_Oscillator/Kinetic_Isotope_Effects (accessed 2024-7-24)

(3) Deuterated Drug. MedChemExpress 2024. https://www.medchemexpress.com/isotope-compound/deuterated-drug.html (accessed 2024-7-24)