A Rapid, Isocratic HPLC Method for Determination of Insulin and Its Degradation Product

This paper aimed to develop a simple, sensitive, and rapid chromatographic procedure for the simultaneous analysis of human insulin and its main decomposition product using isocratic RP-HPLC/UV. A column type RP-C18 (100 × 4.6 mm, 3 μm particle size, and pore size 130 Å) was used. o-Nitrophenol was used as internal standard. The eluent consists of 62% KH2PO4 buffer (0.1 M), 26% ACN, and 12% MeOH. The final pH was adjusted to 3.1. The eluent was pumped at a flow rate of 1.0 mL/min and the effluent was monitored using DAD detector at 214 nm. The method produces a linear response over the concentration range of 0.0106 to 0.6810 mg/mL with detection limit of 0.0029 mg/mL. Considering the specifications of this method, the system was found to be suitable for rapid, direct routine analysis and stability studies of insulin.