The recent development of fused-core technology in HPLC columns is enabling faster and highly efficient separations. This technology was evaluated for the development of a fast analysis method for β-ecdysone in extracts of Pfaffia glomerata. A step-by-step strategy was used to optimize temperature (30–55 °C), flow rate (1.0–2.0 mL min−1), mobile phase composition (mixtures of water and methanol or acetonitrile) and equilibration time (1–5 min). A gradient method has been developed using two solvents: 0.1% acetic acid in water and 0.1% acetic acid in acetonitrile. Optimized conditions provided a method for the separation of β-ecdysone in approximately 2 min with a total analysis time (sample-to-sample) of 9 min, including the return to initial conditions and the re-equilibration of the column. Evaluation of chromatographic performance revealed excellent intraday and interday reproducibility (>99.5%), resolution (2.78), selectivity (1.13), and peak symmetry (1.09) while presenting low limits of detection (0.20 mg L−1) and quantitation (0.67 mg L−1). The robustness of the method has also been calculated according to the concentration/dilution of the sample. Several sample solvents were evaluated and the best chromatographic results were obtained using 80% methanol in water. Finally, the developed method was validated with different extracts of Pfaffia glomerata samples.
