Subcritical Fluid Chromatography at Sub-Ambient Temperatures for the Chiral Resolution of Ketamine Metabolites with Rapid-Onset Antidepressant Effects

Chiral metabolites of ketamine exerting rapid-onset yet sustained antidepressant effects may be marketed directly in the future, but require chemo- and enantio-selective chromatographic methods for quality assurance and control. The chromatographic behavior of S-/R-ketamine, S-/R-norketamine, S-/R-dehydronorketamine, and (2R,6R)-/(2S,6S)-hydroxynorketamine in supercritical fluid chromatography (SFC) was investigated computationally and experimentally with the aim of identifying problematic pairs of enantiomers and parameters for chiral resolution. Retention on three different polysaccharide-based chiral stationary phases (Lux Amylose-2, i-Amylose-3, and i-Cellulose-5) provided new information on the significance of halogen atoms as halogen bond donors and hydrogen bond acceptors for enantioselectivity, which could be corroborated in silico by molecular docking studies. Modifiers inversely affected enantioselectivity and retention. Methanol yielded lower run times but superior chiral resolution compared to 2-propanol. Lower temperatures than those conventionally screened did not impair phase homogeneity but improved enantioresolution, at no cost to reproducibility. Thus, sub-ambient temperature subcritical fluid chromatography (SubFC), essentially low-temperature HPLC with subcritical CO(2), was applied. The optimization of the SubFC method facilitated the chiral separation of ketamine and its metabolites, which was applied in combination with direct injection and online supercritical fluid extraction to determine the purity of pharmaceutical ketamine formulations for proof of concept.