Development, Optimisation, Validation and Inter-Laboratory Verification of a Reversed Phase HPLC Method for Quantification of Human Recombinant Insulin


HPLC methods for insulin in official monographs require extended runtimes and elevated temperatures. Inter-laboratory reproducibility of HPLC methods obtained from published literature is an on-going challenge, moreso for peptides. This paper serves as a step-by step guide to troubleshoot and establish a validated HPLC method for insulin at room temperature using simple UV detectors with minimal run times. A modified gradient reversed-phase HPLC was developed for the quantification of recombinant human insulin with UV detection at room temperature. An octadecylsilica column was used as the stationary phase while the mobile phase consisted of solution A: 1mmol sodium sulphate and 0.2% triethylamine in water and solution B: acetonitrile. The developed method was then validated using International Conference on Harmonisation (ICH) guidelines. The calibration curve was linear over a concentration range of 10-1000 μg/mL with correlation coefficient of 0.9993, with average recovery percent of 100.89 ± 1.4% and RSD recovery of 0.01. Insulin retention time was 3.84 ± 0.08 mins, while LOD and LOQ were estimated at 0.63 and 2.0 μg/mL respectively. The developed method conformed to the validation criteria of the ICH guidelines in our laboratories and other independent operator laboratories, and can serve as a rapid and effective method for quantifying insulin from any sample at room temperature using simple detectors.

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Divisions: College of Health & Life Sciences > Aston Pharmacy School
College of Health & Life Sciences
College of Health & Life Sciences > Chronic and Communicable Conditions
Additional Information: This work is licensed under a Creative Commons Attribution 4.0 International License. Authors retain the copyright of their manuscripts, and all Open Access articles are distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided that the original work is properly cited.
Uncontrolled Keywords: Insulin,HPLC Validation,ICH Guidelines,Inter-Laboratory Reproducibility,Method Development,Optimization,Biotech Products
Publication ISSN: 2348-6201
Last Modified: 27 May 2024 07:23
Date Deposited: 05 Apr 2018 07:35
Full Text Link:
Related URLs: https://rajpub. ... ticle/view/7192 (Publisher URL)
PURE Output Type: Article
Published Date: 2018-03-30
Accepted Date: 2018-03-20
Authors: Iyrie, Affiong (ORCID Profile 0000-0002-2684-2260)
Russell, Craig (ORCID Profile 0000-0003-3791-2161)
Dennision, Tom
Rajoli, Rajith
Saleem, Imran Y.
Rahman, Ayesha S
Mohammed, Afzal (ORCID Profile 0000-0002-5212-3040)



Version: Published Version

License: Creative Commons Attribution

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