Development, Validation and Application of an RP-HPLC Method for the Determination of Reproxalap in Cyclodextrin Inclusion Complexes and an In Situ Ocular Hydrogel.

Background/

Objectives

The aim of this study was to develop and validate a simple, accurate, reproducible, and sensitive reverse-phase high-performance liquid chromatography method for the quantification of reproxalap (RP) in cyclodextrin (CD) inclusion complexes and poloxamer 407 hydrogel formulations.

Methods

Chromatographic separation was achieved using a mobile phase of deionized water and an organic phase (methanol:acetonitrile, 55:45 v/v) in a 40:60 (v/v) ratio, at a flow rate of 0.9 mL/min and a run time of 10 min. Validation assessed linearity, specificity, accuracy, and sensitivity over a concentration range of 1-60 μg/mL. Phase-solubility studies were conducted to determine the apparent stability constant (K1:1), and inclusion complexes were characterized by Fourier transform infrared spectroscopy and differential scanning calorimetry.

Results

The method demonstrated specificity, linearity, sensitivity, and accuracy within the tested concentration range of 1-60 μg/mL. The apparent stability constants (K1:1), calculated from the phase-solubility plot slopes, were 2666.66 M-1 for Sulfobutyl Ether β-Cyclodextrin (SBE-β-CD) and 526.3 M-1 for Hydroxypropyl-β-Cyclodextrin (HP-β-CD). The solubility of RP in deionized water (0.25 mM) increased 8.6-fold to 2.15 mM with the HP-β-CD inclusion complex and 22.4-fold to 5.6 mM with the SBE-β-CD inclusion complex. The method was successfully applied to determine RP in inclusion complexes and poloxamer 407 hydrogel formulations, with no interference from formulation excipients.

Conclusions

The validated RP-HPLC method is suitable for quality control analysis of RP in CD inclusion complexes and poloxamer 407 hydrogel formulations, supporting its application in the future development of these drug delivery systems.