A mixed-mode stationary phase, known as Atlantis BEH C18 AX, offers unique selectivity and was therefore considered as a starting point for devising a method to analyze samples containing analytes with multiple functional groups.
In this application note, we took advantage of the pH stable hybrid silica surfaces and operated the mixed-mode column at pH 6.8, a pH which may lead to degradation if used with traditional silica-based reversed-phase/mixed-mode stationary phases. The Atlantis BEH C18 AX mixed-mode stationary-phase has both C18 and anion-exchange groups and each can contribute to a chromatographic separation. At pH 6.8, the anion-exchange sites were tuned to elute remdesivir triphosphate within the gradient, while the C18 groups played the greater role in the retention of remdesivir. The primary goal of good retention for remdesivir triphosphate and resolution from remdesivir was achieved using these conditions. Additional method optimization could be undertaken to improve the peak symmetry for remdesivir triphosphate, such as reducing mass injected on-column.
Ammonium acetate mobile phases prepared without the use of ion-pair reagents were used in the separation, allowing the option of using optical as well as MS detection. Fast and easy mobile phase preparation was accomplished by using readily available, MS-certified buffer concentrates.
With the emergence of new drug discovery projects, analysts might look to apply this method to in vitro cell studies. Likewise, with further optimization, this technique might prove to be attractive for the analysis of polymerase inhibitors and their metabolites as prepared from biological fluids.