(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

Abstract

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 10min 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 (3.0μm) ODS column (Rs=1.20–4.12). The calibration curves were linear over the concentration range of 1–100μM for each enantiomer of IBU, FLU, and KET, 10–100μM for each enantiomer of NAc-Trp and NAC-Phe, and 50–200μM for each enantiomer of NAc-Leu and NAc-Met. The detection limits (S/N=3) of NSAIDs (1pmol for each enantiomer of IBU, FLU, KET) were one or two orders magnitude lower than that of N-acetylamino acids (10pmol for each NAc-Trp, NAC-Phe enantiomer, 100pmol for each NAc-Leu, NAc-Met enantiomer). The relative standard deviations (RSD, n=5) were less than 3.4% at 60μM for all carboxylic acid enantiomers. The proposed method was applied to the determination of (S)-IBU enantiomer in a pharmaceutical formulation.