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 81
- Downloads 0
A graph-theoretic model of single point mutations in the cystic fibrosis transmembrane conductance regulator
Cystic fibrosis is one of the most prevalent inherited diseases. This disease is caused by a mutation in a membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is known to function as a...
Organic Compounds Generated in Bioethanol Production from Agave Bagasse
In bioethanol production through lignocellulosic residues fermentations are generated by-products such as organic compounds (OCs). The organic compounds (OCs) had been well studied in wine and beer industry, but little i...
Attenuation of DEN induced hepatocellular carcinoma by a novel synthesis of silver nanoparticles using Solanum villosum (Mill.) leaves
Objective: In today’s fast and furious life, the life style and food habits of people are changing drastically, due to which ninety percent of the population suffers with oxidative stress resulting as ageing process, art...
BIOBRAS-16 IMPROVES THE ACCLIMATIZATION OF SPANISH CARNATION (DIANTHUS CARYOPHYLLUS L.) PLANTS OBTAINED IN VITRO
The Spanish carnation (Dianthus caryophyllus L.) is one of the most commercialized flowers worldwide. Its accelerated propagation is possible due to the utilization of micropropagation methods, especially if combined wit...
Impact of ultrasound treatment on molecular structures of casein
Casein was modified by ultrasound treatment (160W, 400 W)and the structural characteristics of casein (control), USC-1 (160W of ultrasound treated casein) and USC-2 (400W of ultrasound treated casein) were investigated....