Evaluation of chiral normal-phase liquid chromatography as a secondary tier in pharmaceutical chiral screening strategy

Abstract

Determination of stereoisomers is an integral part of pharmaceutical analysis. Chiral liquid chromatography (LC) method development is typically initiated through screening of chiral stationary phases (CSPs) and mobile phases (MPs) since chiral separation is difficult to predict. We have previously reported a screening strategy using chiral reversed-phase (RP) LC as two primary tiers due to its versatility for enantio‑recognition and compatibility with diverse sample matrices. Here we focus on developing a normal-phase (NP) LC screening strategy as a secondary tier for chiral method screening. A database was constructed from 60 NPLC screens performed on up to 18 CSPs and 3 MPs using gradient elution. This was used to investigate the effectiveness of NPLC compared to RPLC screening, as well as the impact of MP composition and the selectivity of different CSPs in NPLC screening. A success hit rate of 90% was observed in NPLC compared to 84% in RPLC screening for Bristol Myers Squibb compounds. Importantly, NPLC screening generated successful hit(s) in 81% of the cases that failed in RPLC, demonstrating the value of NPLC as a complementary screening tier. After optimizing the CSP/MP selection, we proposed a NPLC screening workflow with several user-options according to method requirements and instrument capacity. Among these, the most comprehensive NPLC screening consisted of ten CSPs (AD, AS, AY, AZ, OD, OJ, IC, IE, IG, O1) with three MPs. When combined with RPLC, an overall success rate of 97% was achieved for the diverse set of pharmaceutical compounds.