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Updated: Nov 3, 2025

Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns
Published on: April 26, 2016
Chemical derivatization in combination with supercritical fluid chromatography to improve resolution of stereoisomers
Hermes Licea Perez1, Clara Andonian1
1Bioanalysis, Immunogenicity & Biomarkers, In Vivo/In Vitro Translation, GlaxoSmithKline Pharmaceuticals, 1250 S Collegeville Rd, Collegeville, PA 19426, USA.
Abstract:
Aim: Quantification of stereoisomers in biological matrices is of pivotal importance for drug development. Supercritical fluid chromatography paired with chiral stationary phases is the gold standard for resolution of enantiomers. However, this technique often proves inadequate for resolution of polar stereoisomers. Materials & methods: A combination of achiral chemical derivatization with supercritical fluid chromatography using chiral stationary columns to improve enantiomeric resolution is described. Results: Separation of four possible stereoisomers of linerixibat was achieved after derivatization with 3N HCl in n-butanol within 12 min (case1). Derivatization with acetic, propionic, butyric, isobutyric, valeric and isovaleric anhydrides significantly improved the separation of stereoisomers (case 2 and 3) within 10 min. The best stereoisomeric resolution was achieved using valeric and isovaleric anhydrides.
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