Verubecestat is an inhibitor of β-site amyloid precursor protein cleaving enzyme 
1 (BACE1) being evaluated in clinical trials for the treatment of Alzheimer's 
disease. Synthetic route development involves diastereoselective transformations 
with a need for enantiomeric excess (ee) determination of each intermediate and 
final active pharmaceutical ingredient (API). The analytical technical package 
of validated methods relies on enantioselective SFC and RPLC separations using 
multiple 3 and 5 μm coated polysaccharide-based chiral stationary phases (CSPs) 
and mobile phases combinations. Evaluation of recently developed chiral columns 
revealed a single chiral selector (Teicoplanin) bonded to 2.7 μm core-shell 
particles using H3PO4 in H2O/ACN and triethylammonium acetate: methanol based 
eluents at different isocratic compositions allowed good enatioseparation of all 
verubecestat intermediates. EE determination of verubecestat is easily performed 
on NicoShell, another macrocyclic glycopeptide chiral selector bonded to 2.7 μm 
superficially porous particles. This approach enables fast and reliable 
enantiopurity analysis of the entire verubecestat synthetic route using only two 
chiral columns and mobile phases on a conventional HPLC system, simplifying 
technical package preparation, method validation and transfer to manufacturing 
facilities.