Determination of total and encapsulated insulin in a vesicle formulation.

A method for the determination of the amount of insulin in a vesicle formulation was developed. Samples were treated with anion exchange resin to quantitatively remove the insulin outside the vesicle walls. Encapsulated insulin was released from vesicles by disruption with a surfactant and the amount released was determined by reversed-phase HPLC. Recovery of insulin from the vesicle matrix was 99, 97, and 98% for vesicle solutions spiked with 1.0, 0.5, and 0.2 U/mL of insulin, respectively. The sample preparation steps resulted in removal of greater than 99.5% of the unencapsulated insulin; 98% recovery of the vesicles from the resin; 97% recovery of the encapsulated insulin from the resin; and greater than 99% disruption of the vesicles by the surfactant. Precision of the measurements for the amounts of total and encapsulated insulin was 2.7 and 3.3% relative standard deviations, respectively, for insulin levels of 0.7 U/mL.