Feed injection-enabled reversed phase liquid chromatography for simplified analysis of lipophilic drugs and formulations.

Strong sample solvents and lipidic excipients hamper reversed‑phase (RP) liquid chromatography (LC) analysis of lipid‑based pharmaceutical formulations by causing solvent mismatch, resulting in peak distortion and restricting the applicable injection volume, which ultimately reduces the sensitivity to detect impurities. In this work, we evaluated feed injection (FI) under gradient conditions for the analysis of sesame oil-based formulations dissolved in ethyl acetate (EtOAc) and integrated FI with two‑dimensional LC coupled to mass spectrometry (2D‑LC/MS) for impurity profiling. Findings were compared to conventional flow‑through injection (FTI) and sandwich injection, across injection volumes of 1-40 µL. FI at low feed flow fractions (≤ 5 % of mobile phase flow rate) provided ≥ 1:20 online dilution, sustaining column head focusing, and preserving peak shape and linearity across the full injection range. In contrast, higher feed flow fractions (≥ 20 % of the mobile phase flow rate, ≤ 1:5 dilution) resulted in severe fronting peaks and non-linear ultraviolet (UV) detector response. Finally, the 2D transfers resolved matrix‑ and Active Pharmaceutical Ingredient (API)‑related impurities without laborious offline reconstitution or dilution. This work extends the application field of FI from analyte diluents to complex lipid matrices, offering a robust, scalable workflow for pharmaceutical formulation analysis.