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

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

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

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Publisher
The Royal Society of Chemistry
Copyright
This journal is © The Royal Society of Chemistry
ISSN
1759-9660
eISSN
1759-9679
D.O.I.
10.1039/c8ay00502h
Publisher site
See Article on Publisher Site

Abstract

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

Journal

Analytical MethodsRoyal Society of Chemistry

Published: May 17, 2018

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