Methotrexate (MTX) is a derivative of aminopterin, used as an anticancer or an anti-inflammatory agent. The development of suitable drug delivery systems containing MTX is an active area of research, requiring suitable analytical methods. Therefore, a high-throughput HPLC method is proposed for determination of MTX in the delivery system and permeation studies. Chromatographic separation was achieved on a reversed phase monolithic C18 column using isocratic elution (phosphate buffer (pH 7.0, 10 mM)-ACN (91:9, v/v)) and spectrophotometric detection at 302 nm. Total run time was 3.5 min, with MTX retention time of 2.1 min, providing 17 determinations per hour. The method was found to be specific, accurate (99.2–110%) and precise for intra-day (RSD ≤ 3.5%) and inter-day assays (RSD ≤ 3.4%). MTX showed stability after 24 h at room temperature or in the autosampler (4 °C) and over three freeze-thaw cycles with recoveries ≥94.2%. The validated method was successfully applied to establish in vitro drug release profile of MTX delivered by lipid nanoparticles. Application to pig skin permeation media provided mean recovery values ranging from 94.1 to 101.6% (RSD ≤ 1.1%).
- Monolithic column
- Skin surrogate