Determination of Insulin in Rabbit Plasma Using High-Performance Liquid Chromatography

The aim of the present study was to develop and validate a procedure based on high-performance liquid chromatography (HPLC) for determination of insulin in rabbit plasma. Separation of insulin was achieved on an Ace C18 column (5 mu m, 250x4.6 mm i.d.) using diode array detection (DAD) with lambda = 206 nm. The mobile phase consisted of 0.2 M sulfate buffer (pH 2.4)-acetonitrile (75: 25, v/v). The analysis was performed in less than 15 min with a flow rate of 1.2 mL/min. Excellent linearity was found between 0.15 and 10 mu g/mL. Intra-and inter-day precision values for insulin in plasma were less than 10.2, and accuracy (relative error) was better than 12.4%. The recoveries for all samples were > 86.7%. The limits of detection (LOD) and quantification (LOQ) of insulin were 0.10 and 0.15 mu g/mL, respectively. The described HPLC method has adequate sensitivity and specificity to study pharmacokinetics of insulin in rabbits, and could be adapted also to clinical pharmacokinetic study.