Development, optimization and validation of a sub-minute analytical enantioselective high performance liquid chromatographic separation for a folic acid precursor in normal phase mode

A sub-minute enantioselective normal phase high performance liquid chromatographic (HPLC) method for the analysis of a chiral precursor molecule employed frequently in folic acid syntheses was developed, optimized and successfully validated according to ICH-guidelines. It could be shown that ultra-high performance chromatography (UHPLC) can give significant advantages compared to traditional HPLC not only in reversed phase HPLC, but also for enantioselective separations in normal phase mode. Novel 3 μm-particle sizes allow developing analytical chromatographic methods completely resolving two enantiomers in the shortest time possible while preserving high efficiency and low detection limits. By offering increased resolution, sensitivity and speed, enantioselective UHPLC (eUHPLC) improves sample throughput, productivity and provides considerably faster access to information on enantiomeric purity also under non-aqueous conditions.

► Validated sub-minute enantioselective NP HPLC method replaces polarimetric determination. ► First enantiomer separations of p-amino-benzoyl-(D/l)-(−)-glutamic acid. ► Faster access to information on enantiomeric purity under non-aqueous conditions. ► Routine QC work on small particle size col. needs ∼1/4 of solv. and is 7.5 times faster. ► High precision even for samples cont. only 0.01% of non desired enantiomer (ee 99.98%).