Synthesis and evaluation of a novel chiral derivatization reagent for resolution of carboxylic acid enantiomers by RP-HPLC

A novel derivatization reagent, N-[1-Oxo-5-(triphenylphosphonium)pentyl]-(S)-3-aminopyrrolidine (OTPA), with triphenylphosphine (TPP) as a basic structure carrying a permanent positive charge was developed for the enantiomeric separation of chiral carboxylic acids by high-performance liquid chromatography (HPLC). OTPA reacted with the carboxylic acids at 40 °C within 90 min in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 1-hydroxybenzotriazole (HOBt). The degree of epimerization (racemization) during the derivatization reaction was negligible. The separability of the diastereomers was evaluated in terms of their resolution value (Rs). The Rs values of the derivatives of non-steroidal anti-inflammatory drugs (NSAIDs), which were selected as the representative carboxylic acids, were completely separated by reversed-phase chromatography using an ODS (4.6 mm × 150 mm I.D., 5.0 μm) column. The resolution Rs values were 1.54-2.23 for the evaluated carboxylic acids and the OTPA-derivatization was also effective for the enantiomeric separation of chiral carboxylic acids. The calibration curves (r2 > 0.9971) were linear over the concentration range of 0.0125-1.25 mM for each enantiomer of ketoprofen (KET), and naproxen (NAP), 0.05-1.0 mM for each enantiomer of ibuprofen (IBU), 2-phenylpropionic acid (PPA), and loxoprofen (LOX), and 0.05-1.25 mM for each enantiomer of PBA. The limit of detection (S/N = 3) for each of the enantiomers of the NSAIDs and chiral carboxylic acid enantiomers was 1.4-7.6 μmol/L. The inter-day and intra-day assay precisions were all < 6.77% and the mean recoveries (%) of the NSAIDs and chiral carboxylic acids from the spiked human plasma were 95.27-101.12%. The derivatization followed by HPLC-UV enabled the separation and detection of NAP in human plasma with simple pretreatment.