Insights into the separation, enantioseparation and recognition mechanisms of methamphetamine isotopologues on achiral and polysaccharide-based chiral columns in high-performance liquid chromatography

Abstract

Background

Isotopologues resulting from the labelling of molecules with deuterium have attracted interest due to the isotope effect observed in chemistry and biosciences. Isotope effect may also play out in noncovalent interactions and mechanisms leading to intermolecular recognition. In chromatography, differences in retention time between isotopologues, as well as between isotopomers have been observed resulting in two different elution sequences (isotope effects): the normal isotope effect when heavier isotopologues retain longer than lighter analogues, and the inverse isotope effect featuring the opposite elution order. Although several cases of deuterium isotope effects have been reported so far, the molecular bases of these phenomena remain unclear.

Results

We report the separation of isotopologues of methamphetamine (N-methyl-1-phenylpropan-2-amine) (MET), featuring different number and location of deuterium substituents, on an achiral column by high-performance liquid chromatography (HPLC), as well as the simultaneous separation of isotopologues and their enantiomers on some polysaccharide-based chiral columns. The effects of the number and location of deuterium substituents introduced in MET’s structure, of the surface chemistry of the adsorbent, and the mobile phase composition and pH on the retention and separation of isotopologues and their enantiomers were examined. In several cases, the effect of temperature was also evaluated, and the thermodynamic quantities associated with isotopologue adsorption, separation, and enantioseparation were also calculated. To elucidate the molecular bases of the experimental observations, quantum mechanics calculations were performed focusing on the vibrational degree of freedom calculated for models of isotopologue-selector complexes. On this basis, zero-point vibrational energies were computed as useful descriptors to differentiate computationally between deuterated isotopologues.

Significance

Using HPLC as experimental technique with the following dual function: 1) separation of MET isotopologues and their enantiomers; 2) exploring noncovalent interactions underlying their separation. The degree of deuteration, location of deuterium substituents, and mobile phase pH play key roles in isotopologue separations. By integrating experimental and computational analyses, noncovalent interactions underlying normal and inverse isotope effects were deconvoluted, highlighting the contribution of hydrogen bond, hydrophobic and dispersive forces in isotopologue and enantiomer separation.