Development of novel enantioselective HPLC methods for the determination of the optical purity of Nα-Fmoc/Boc amino acid derivatives (Nα-PADs) of natural and unnatural amino acids: an analytical strategy for the key starting materials of therapeutic peptides

作者: Yagnakirankumar Komaravolu , Venugopala Rao Dama , Thirumala Chary Maringanti

DOI: 10.1039/C8AY00502H

关键词: Ion exchangeStereochemistryEnantiomerSolventSelectivityEnantiomeric excessChiral column chromatographyChemistryAmino acidDiastereomer

摘要: Control of the chiral purity Nα-Fmoc/Boc amino acid derivatives (Nα-PADs) used in manufacturing therapeutic peptides is an important material quality attribute to reduce risk undesired pharmacological and toxicological effects arising from related diastereomeric impurities drug substances. In this study, HPLC methods for 4 pairs unnatural (Tic, Oic, Ala(thi) Hyp) 11 natural Nα-Fmoc were developed optimized on carbohydrate-based stationary phases (CSPs) – CHIRALPAK IA, IC QNAX. Mobile phase solvent polarity played a major role recognition events using CSPs, among other method parameters such as temperature, flow rate mobile additives. QNAX able show all D L enantiomers Nα-PADs, with better selectivity (1.32 < α 2.12) resolution (2.9 Rs 7.7) towards CSP. Natural Nα-PADs also showed (1.18 2.88) (2.2 12.5) CSP compared (1.02 1.59) (1.6 6.0), respectively. Enantiomeric Phe was higher IA (1.64 2.67; 7.41 8.62) CSPs. Considering trends hydrophobicity (log P) obtained separation factors (α), especially primarily influenced by (π–π interactions) due protecting groups when analysed under similar mixtures chromatographic conditions. The can be ion exchange dipole–dipole interactions evident lower retention times both Nα-PADs. These further validated per ICH guidelines sensitive estimating isomers (≤0.05%) presence desired isomers.

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