Stability-indicating RP-HPLC method for simultaneous determination of formoterol fumarate, glycopyrrolate, and fluticasone propionate in pharmaceutical formulations

A stability-indicating reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the simultaneous quantification of Formoterol Fumarate (FOR), Glycopyrrolate (GLY), and Fluticasone Propionate (FLU) in triple fixed-dose inhalation formulations. Utilizing a C18 column (300 mm × 3.9 mm, 4 μm) with gradient elution (0.1% formic acid in water and acetonitrile: methanol [1:1] with 0.1% formic acid, flow rate 1.0 mL/min), the method achieved excellent peak resolution at retention times of 3.206 min (GLY), 6.882 min (FOR), and 21.960 min (FLU). Optimization via a Box–Behnken Design within an AQbD framework ensured robustness, yielding a composite desirability of 1.000. The method exhibited linearity (r² ≥ 0.9999) across 5–25 µg/mL for FOR and GLY, and 120–360 µg/mL for FLU, with limits of detection (LOD) of 0.110–1.454 µg/mL and limits of quantification (LOQ) of 0.332–4.406 µg/mL. Precision (%RSD < 1.6%) and accuracy (recovery 98.5–101.2%) were exceptional, with system suitability parameters (tailing factor < 2.0, theoretical plates > 18,000, resolution > 26) meeting ICH Q2(R1) standards. Forced degradation studies under ICH Q1A(R2) conditions revealed maximum degradation under oxidative stress (FOR: 18.86%, GLY: 14.88%, FLU: 15.57%), with degradation products well-resolved from parent peaks and mass balance exceeding 80%. Greenness evaluation using the AGREE metric (score: 0.71) and White Analytical Chemistry framework confirmed environmental sustainability and analytical efficiency. This robust, sensitive, and eco-friendly method is ideally suited for routine quality control and regulatory assessment of triple-combination inhalers, advancing sustainable pharmaceutical analysis.