Enantiomeric analysis and separation of three chiral carboxylic acids via derivatization using chiral pure derivative

Abstract An indirect high-performance liquid chromatographic method was developed for the enantiomeric analysis of pharmaceutically relevant chiral carboxylic acids, including (±)-2-fluoro-α-methyl-4-biphenylacetic acid, (±)-2-(10,11-dihydro-10-oxodibenzo[b,f]thiepin-2-yl)propionic acid, and (±)-2-(3-phenoxyphenyl)propionic acid The enantiomeric analytes were converted into diastereomeric anhydrides via activation of the carboxyl group with N-hydroxybenzotriazole in the presence of dicyclohexylcarbodiimide and 4-dimethylaminopyridine, employing a twofold molar excess of the chiral derivatizing reagent to ensure complete derivatization without kinetic resolution. Chromatographic separation was achieved on a reversed-phase column using triethylammonium phosphate buffer (10 mM, pH 3.5) with acetonitrile as organic modifier; a linear gradient of 25–75 % acetonitrile over 45 min at 1.0 mL min −1 afforded baseline resolution, improved peak symmetry, and reduced analysis time compared to methanol-based systems. The method exhibited excellent linearity over the range 25–250 ng mL −1 (R 2 ≥ 0.97), with satisfactory precision (RSD 2.6–5.4 %) and accuracy (recoveries 89.5–101.6 %). The developed method is well suited for routine enantiomeric purity assessment of chiral carboxylic acid drug candidates.