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Novel, efficient, facile, and comprehensive protocol for post-column amino acid analysis of icatibant acetate containing natural and unnatural amino acids using the QbD approach

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Qualitative and quantitative determination of amino acid composition using amino acid analysis (AAA) is an important quality attribute and considered an identity of therapeutic peptide drugs by the regulatory agencies. Although huge literature is available on pre- and post- column derivatization AAA methods, arriving at an appropriate hydrolysis protocol coupled with adequate separation of the derivatized/underivatized amino acids is always challenging. Towards achieving a facile and comprehensive protocol for AAA, the present work is geared towards developing a deeper understanding of the extent of hydrolysis of peptide, and the nature and stability of amino acids present in the peptide backbone. This defines the suitability of the method in meeting the end goals and the regulatory requirement. Analysis of historical data generated during the method optimization of AAA for icatibant acetate (ICT) using head space oven hydrolysis (HSOH) and microwave-assisted hydrolysis (MAH) methods helped in arriving at fast (< 1 h) and efficient hydrolysis (0.9–1.1 of theoretical residue) conditions. Better separations for the natural and unnatural amino acids were achieved using 3.45 ≤ pH ≤ 10.85, and a column oven gradient program. This approach was useful in meeting the method quality attributes [resolution (Rs) > 2.0; plate count (N) > 5600; and USP tailing factor < 1.2] with a target analytical method profile of relative amino acid mole ratios (RAAMR) in the range of 0.9–1.1 for Ser, Oic, Tic, Hyp, Ala (Thi), Gly and Pro, and between 2.7 and 3.3 for Arg. The developed method was validated as per the ICH guidelines and is precise, accurate, linear and robust.

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Amino acid analysis


Icatibant acetate


Head space oven hydrolysis


Microwave-assisted hydrolysis


Relative amino acid mole ratios

R s :



United States pharmacopeia














Octahydro-1H-indole-2-carboxylic acid


D-1, 2, 3, 4-Tetrahydroisoquinoline-3-carboxylic acid


Therapeutic peptides


Nuclear magnetic resonance spectroscopy


International Union of Pure and Applied Chemists


Committee for Medicinal Products for Human Use


Pre-column derivatization


Post-column derivatization


Response surface methodology








Peak tailing




Theoretical plate count


Figure of merit


Extent of hydrolysis


Head space gas chromatography


Gas chromatography


International Conference on Harmonization


Relative standard deviation


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Two of the authors (KYKK and DVGR) thank the management of Dr. Reddy’s Laboratories Ltd., Hyderabad, India, for supporting this work. Support from colleagues in Research and Development, and Analytical Research and Development of Dr. Reddy’s Laboratories Ltd. is appreciated.

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Correspondence to Yagnakirankumar Komaravolu.

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The authors Yagnakirankumar Komaravolu, Venugopala Rao Dama, and Thirumala Chary Maringanti declare that they have no conflicts of interest.

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Komaravolu, Y., Dama, V.R. & Maringanti, T.C. Novel, efficient, facile, and comprehensive protocol for post-column amino acid analysis of icatibant acetate containing natural and unnatural amino acids using the QbD approach. Amino Acids 51, 295–309 (2019).

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