Assessing extreme enantiomeric purity of (+)-1-(9-Fluorenyl)ethyl chloroformate by normal- and reversed-phase enantioselective chromatography: toward greener analytical strategies.

FLEC (1-(9-fluorenyl)ethyl chloroformate) is widely used as a chiral derivatizing agent (CDA) in indirect enantioselective chromatography, where its enantiomeric purity critically affects quantitative accuracy. Even trace contamination with the opposite enantiomer can generate additional diastereoisomers and introduce systematic bias in extreme enantiomer determination. In this study, normal-phase (NP) and reversed-phase (RP) enantioselective HPLC and UHPLC methods were evaluated to assess the extreme enantiomeric purity of (+)-FLEC after hydrolysis under pharmacopoeial conditions. NP-UHPLC on Pirkle-type stationary phases enabled the application of the Inverted Chirality Column Approach (ICCA), achieving high-resolution separations (Rs up to 5.90) and accurate determination of the minor enantiomer (98.86% ee). RP chromatography on polysaccharide-based stationary phases allowed direct analysis of aqueous samples. A ternary mobile phase (water/methanol/isopropanol, 80:10:10) improved resolution (Rs 2.80 vs 1.79), reduced analysis time (×2.2), and lowered solvent consumption (∼50%) compared to a binary water/acetonitrile system. The minor enantiomer content was determined as 0.43% versus 0.40%. Although the absolute difference is small (0.03%), it leads to a relative deviation of up to 15% when propagated through the USP correction model near regulatory limits. Overall, RP provides a reliable and greener alternative, while NP-UHPLC remains the reference benchmark for extreme enantiomeric excess determination.