Isolation and purification of β-sitosterol from avocado ( Persea americana Mill.) seed extract using a multi-step chromatographic approach

Avocado (Persea americana Mill.) seeds are an abundant, largely-discarded fruit-processing by-product rich in bioactive phytosterols. This study developed a systematic, scalable protocol for valorising this waste by isolating high-purity β-sitosterol. Dried seeds of the Minion variety were extracted by maceration with methanol, and the crude extract was fractionated by vacuum liquid chromatography (VLC) using step-gradient elution; the procedure was optimised on a small scale and transferred to production scale. The β-sitosterol-rich fraction was then purified by a two-stage flash-chromatography process combining normal- and reversed-phase modes. From 177.9 g of this fraction, 40.5 g of purified compound was obtained over fifteen reproducible reversed-phase runs. The isolate gave a single thin-layer chromatography (TLC) spot and a single high-performance liquid chromatography (HPLC) peak (∼11.2 min) matching an authentic standard, with nuclear magnetic resonance (NMR) signals fully consistent with reported β-sitosterol data. The approach offers an effective, reproducible, and scalable route for valorising avocado-seed waste into a high-value phytosterol.