Development and validation of a gradient HPLC method for the determination of insulin in rat plasma: application to pharmacokinetic study / Eda Frida Salleh

A simple, specific and sensitive reversed-phase high performance liquid chromatography method was developed to determine insulin in rat plasma. The method involves direct injection of rat plasma sample after deproteinization using acetonitrile - propanol (1:1). The mobile phase comprises a mixture of water and acetonitrile containing 0.1% v/v trifloroacetic acid (TFA) with gradient analysis by increasing the proportion of mobile phase of water from 25% v/v to 40% v/v within 20 minutes. A Phenomenon C18 column (5μm 4.6 x 150 mm) was used for the chromatographic separation which was delivered at a flow rate of 0.6 mL/min at 210 nm wavelength. Under these conditions, the method was validated with respect to linearity, recovery, specificity, accuracy, precision, and stability. The method was proven to be linear over the concentration range of 0.39 - 50.00 μg/ml with mean correlation coefficient of 0.9994. The mean extraction recovery was 97.4%, while the precision of within-day and between-day measurements was a <9%. The limit of detection (LOD) and limit of quantitation (LOQ) of the method were 0.13 μg/ml and 0.39 μg/ml, respectively. The short run time, specificity, sensitivity and reproducibility of this method were found to be satisfactory and thus suitable for routine determination of insulin in rat.