Pharmaceutical development and stability-indicating high-performance liquid chromatography characterization of an insulin ophthalmic formulation for the treatment of persistent corneal epithelial defects

Objectives
Persistent corneal epithelial defects remain a challenging ocular condition, and topical insulin has emerged as a promising therapeutic approach to promote corneal epithelial healing. However, the pharmaceutical development of compounded insulin ophthalmic formulations remains insufficiently standardized. This study aimed to develop a preservative-free insulin-based ophthalmic formulation suitable for pharmaceutical compounding and to establish a validated stability-indicating analytical method for insulin quantification.

Methods
Three commercial insulin preparations were first screened as potential sources of active pharmaceutical ingredient for ophthalmic compounding. Four excipient formulations were subsequently investigated during a rational preformulation screening. Physicochemical characteristics were assessed. Insulin glulisine (IG) was selected for further development. A reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated. Specificity, linearity, trueness, precision, detection and quantification limits were evaluated. The stability-indicating capability of the method was assessed through forced degradation under acidic, alkaline, oxidative, thermal and photolytic conditions.

Results
IG formulated in 0.9% sodium chloride with 1.5 mg/mL sodium hyaluronate was selected based on its favourable physicochemical characteristics and chromatographic compatibility. The formulation remained clear and showed near-physiological pH and osmolality. The RP-HPLC method demonstrated adequate specificity and was validated over the 0.16–6.40 IU/mL range. Relative biases ranged from +0.31% to +0.71%, while repeatability and intermediate precision coefficients of variation ranged from 0.10% to 1.77% and 0.96% to 1.81%, respectively. The estimated detection and quantification limits were 0.046 and 0.152 IU/mL. Forced degradation studies demonstrated that the IG peak area decreased by 63.0% after 1 h in 0.5 M NaOH and became undetectable after 4 h. Thermal stress at 60 °C resulted in an 86.1% decrease in the insulin peak area after 24 h, while ultraviolet exposure caused a 38.1% decrease after 24 h.

Conclusions
A preservative-free ophthalmic formulation of IG containing sodium hyaluronate was successfully developed through a rational pharmaceutical approach following the screening of commercial insulin sources and excipient combinations. The validated RP-HPLC method demonstrated suitable analytical performance and stability-indicating capability, providing a reliable tool for monitoring insulin integrity during subsequent stability studies. This formulation and analytical strategy provide a basis for further evaluation of the long-term physicochemical and microbiological stability of compounded insulin eye drops.

Key words: insulin eye drop, RP-HPLC, persistent corneal epithelial defects