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

  • Affiong Iyire Aston University, Birmingham
  • Craig Russell Aston University
  • Tom Dennison Malvern Panalytical Ltd
  • Rajith Rajoli Liverpool John Moores University
  • Imran Saleem Liverpool John Moores University
  • Ayesha Rahman University of Wolverhampton
  • Afzal Mohammed Aston University
Keywords: Insulin, HPLC Validation, ICH Guidelines, Inter-Laboratory Reproducibility, Method Development, Optimization, Biotech Products


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.

Author Biographies

Affiong Iyire, Aston University, Birmingham

Aston Pharmacy School, Aston University, Birmingham, UK, B4 7ET.

Craig Russell, Aston University

Aston Pharmacy School, Aston University, Birmingham, UK, B4 7ET.

Tom Dennison, Malvern Panalytical Ltd

Malvern Panalytical Ltd, Grovewood Road, Malvern, UK, WR14 1XZ.

Rajith Rajoli, Liverpool John Moores University

School of Pharmacy & Biomedical Sciences, Liverpool John Moores University, Liverpool, L3 3AF

Imran Saleem, Liverpool John Moores University

School of Pharmacy & Biomedical Sciences, Liverpool John Moores University, Liverpool, L3 3AF.

Ayesha Rahman, University of Wolverhampton

School of Pharmacy, Faculty of Science & Engineering, University of Wolverhampton, UK, W1 1QU.

Afzal Mohammed, Aston University

Aston Pharmacy School, Aston University, Birmingham, UK, B4 7ET.


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