%0 Journal Article
%T A Rapid, Isocratic HPLC Method for Determination of Insulin and Its Degradation Product
%A Ahmad Najjar
%A Mahmoud Alawi
%A Najiah AbuHeshmeh
%A Alsayed Sallam
%J Advances in Pharmaceutics
%D 2014
%R 10.1155/2014/749823
%X 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 ¡Á 4.6£¿mm, 3£¿¦Ìm particle size, and pore size 130£¿£¿) 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. 1. Introduction Since insulin was discovered in 1921 by Frederick Banting and Charles Best, it has become one of the most thoroughly studied molecules in scientific history. Insulin is a pancreatic hormone that treats diabetes by controlling the amount of sugar in the blood. This peptide hormone is composed of 51 amino acids distributed between two peptides chains, one comprising 21 amino acids (chain A) and the other 30 amino acids (chain B). The two chains are joined together by two disulfide bonds between two cysteine residues, where a disulfide bond is the linkage bond between two sulfurs [1]. Like most proteins, insulin is not stable in aqueous solutions. Its degradation mainly goes through hydrolytic reactions (deamidation) or polymerization mechanisms. Deamidation at asparagine amino acid in chain A of insulin (A21) is considered as the main degradation product of insulin at low pH values, while in neutral or alkaline medium, deamidation at asparagine B3 occurs [2]. Polymerization may also occur in neutral and alkaline medium through transamidation reactions [3]. As a diabetes medicine, the purity of recombinant insulin must be more than 98%, with the insulin-related compound A21 desamido insulin being less than 2% [4, 5]. Most commercial insulin preparations are containing low concentrations of phenol and/or m-cresol as preservatives from bacterial contaminations. Separation of insulin and its related compounds is a difficult mission due to the insignificant differences in chemical structures. Many immune and nonimmune methods have been reported for determination of human insulin. Radioimmunoassay [6¨C9], enzyme immunoassay [10¨C13], luminescent immunoassay [14], capillary electrophoresis [15¨C17], and high performance liquid
%U http://www.hindawi.com/journals/ap/2014/749823/