Development of Analysis and Validation of Human Insulin-Containing Matrices Using Reverse Phase-Liquid Chromatography

Licensed drugs containing human insulin are used as a routine therapy to maintain blood glucose levels in patients with diabetes mellitus. Meanwhile, production and storage factors can reduce the potency of these drugs, and this is important for patients. Therefore, a fast and reliable analytical method is needed to ensure quality, as well as safety and effectiveness without being affected by the matrices content, such as m-cresol and glycerol. The study was developed with the high-performance liquid chromatography using a reverse-phase column ReliantÔ C-18 (4.6 x 150 mm, 5 mm), 40°C column temperature, 215 nm UV-Vis detector, 1 mL/min flow rate, and 20 mL injection volume, while, ethylparaben was used as an internal standard. Furthermore, the separation was conducted with solution A and B containing Na2SO4 pH 2.3 and Na2SO4 pH 2.3 in acetonitrile (55:45, v/v) in a ratio of 38:62, v/v, while validation was performed according to ICH guideline. The human insulin retention time was 5.04±0.009 minutes, with linearity of 9.74-146.03 mg/mL (r = 0.9997). Accuracy and precision were 100.71±1.11% and 0.64%, while LOD and LOQ were 0.193 mg/mL and 0.643 mg/mL respectively. Human insulin remained stable at 23°C for 48 hours as demonstrated by ANOVA, a0.05. Based on the results, this method has the potential to separate human insulin from A-21 desamido insulin as a degradant and matrices, as well as allowing for testing many products in a short time.