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Method Development and Validation of Insulin Estimation in Insulin Degrading Enzyme Assay using RP-HPLC

Vinyas Mayasa, Vijay Kumar P. Rasal and Banappa S. Unger

Insulin Degrading Enzyme (IDE) highly conserved zinc metallopeptidase is the primarily responsible for high specific degradation of insulin. The aim of this project is to develop a simple, sensitive, and rapid chromatographic procedure for the analysis of human insulin remaining in Insulin Degrading Enzyme assay using isocratic RP-HPLC/UV. A column type RP-C18 (150 × 4.6 mm, 5 􀟤m particle size). The eluent consists of 50% sodium sulfate (0.2 M, pH 3.1 adjusted with o-phosphoric acid), 50% ACN. The eluent was pumped at a flow rate of 1.0 mL/min and the effluent was monitored using UV detector at 214 nm. The method produces a linear response over the concentration range of 0.15-25 μg/mL of insulin and linear as well with increase in the protein concentration. The method is validated for specificity, linearity, precision, accuracy and limit of detection and quantification. Also, the method developed can be applied for the estimation of insulin in the IDE assay.