(S)-1-methyl-4-(5-(3-aminopyrrolidin-1-yl)-2,4-dinitrophenyl)piperazine as a novel chiral derivatization reagent for high-performance liquid chromatographic analysis of carboxylic acid enantiomers

A novel chiral derivatization reagent, (S)-1-methyl-4-(5-(3-aminopyrrolidin-1-yl)-2,4-dinitro-phenyl)piperazine (APy-PPZ) was developed for the enantiospecific determination of chiral carboxylic acids in high-performance liquid chromatography. APy-PPZ reacted with carboxylic acids within 10 min at 30 °C in the presence of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM). Epimerization during the derivatization reaction was negligible. Diastereomers derived from non-steroidal anti-inflammatory drugs (NSAIDs) and N-acetylamino acids were completely separated by reversed-phase chromatography using a small particle (3.0 μm) ODS column (Rs = 1.20-4.12). The calibration curves were linear over the concentration range of 1-100 μM for each enantiomer of IBU, FLU, and KET, 10-100 μM for each enantiomer of NAc-Trp and NAC-Phe, and 50-200 μM for each enantiomer of NAc-Leu and NAc-Met. The detection limits (S/N = 3) of NSAIDs (1 pmol for each enantiomer of IBU, FLU, KET) were one or two orders magnitude lower than that of N-acetylamino acids (10 pmol for each NAc-Trp, NAC-Phe enantiomer, 100 pmol for each NAc-Leu, NAc-Met enantiomer). The relative standard deviations (RSD, n = 5) were less than 3.4% at 60 μM for all carboxylic acid enantiomers. The proposed method was applied to the determination of (S)-IBU enantiomer in a pharmaceutical formulation.