Development, Optimisation, Validation and Inter-Laboratory Verification of a Reversed Phase HPLC Method for Quantification of Human Recombinant Insulin
Journal Title: JOURNAL OF ADVANCES IN BIOTECHNOLOGY - Year 2018, Vol 7, Issue 0
Abstract
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.
Authors and Affiliations
Affiong Iyire, Craig Russell, Tom Dennison, Rajith Rajoli, Imran Saleem, Ayesha Rahman, Afzal Mohammed
Keywords
Related Articles
- EP ID EP653907
- DOI 10.24297/jbt.v7i1.7192
- Views 54
- Downloads 0
Cranberry Extract Suppressive Effect on Secretion of Inflammatory Cytokines by the Monocytic Cells
The proanthocyanidin rich fraction of cranberry (NDM) has long been speculated to possess the capacity to suppress the production of inflammatory mediators by macrophages. The objective of this investigation was to exami...
Polyphasic Characterization of a Potential Novel Cellulolytic Bacterium Brevibacillus Brevis Strain St-2
The tremendous commercial potential of cellulases in a variety of applications remains the driving force for research in this area. The present study was aimed at isolation and screening of promising cellulolytic strains...
2G Ethanol Production From Palm Lignocellulosic Biomass
Brazil presents the world's largest potential for the production of palm oil due to nearly 75 million hectares of land suitable for palm culture and advantageous soil and climate. The biomass generated in the pro...
In silico analysis to elect superior bacterial alkaline protease for detergent and leather industries
Alkaline protease contributes 40% of the total worldwide enzyme sales. Alkaline protease that is stable at very high temperature and pH is massively desirable for detergent industry and leather industry specially in tann...
Extraction and Purification of Antimicrobial Proteins from Datura Stramonium Seed
Resistant to antibiotics by microbes have become a major global challenge incurring economic and public health burden. Hence, research to develop new effective anti-biotics should be a major consideration to pharmaceutic...