Fast and green sterols analysis by microwave-assisted saponification and extraction followed by solid phase extraction.

Sterol analysis is routinely conducted in many laboratories, as sterols are key markers for detecting adulteration in extra virgin olive oil (EVOO). Given EVOO's high nutritional and economic value, it is often subject to fraud, typically through blending with lower-cost oils. Accurate sterol profiling is therefore essential for authenticity verification and regulatory compliance.
The official analytical method involves several labor-intensive steps: saponification, liquid-liquid extraction, thin-layer chromatography (TLC), and gas chromatography with flame ionization detection (GC-FID) [1]. While effective, this workflow is time-consuming (approximately five hours), requires large volumes of organic solvents, and involves significant manual handling. These limitations reduce its applicability for high-throughput analysis and does not align with the principles of green chemistry [2]. To address these issues, the aim of this work is to optimize the official method and to have faster and greener process that ensuring performance levels that are fully comparable to those of the official method.
The method that has been developed combines saponification and liquid-liquid extraction into a single microwave-assisted saponification and extraction (MASE) step, which significantly reduces the processing time and solvent amount. In addition, the step of TLC purification has been replaced by solid-phase extraction (SPE) purification that has been optimized by adjusting solvent composition to improve sterol separation while further decreasing solvent consumption.
The greenness of the methods was assessed using the AGREEPrep software, confirming superior sustainability performance compared to the conventional method.
Overall, the optimized protocol reduces total analysis, and supports both quality assurance and the principles of green analytical. This makes it a valuable alternative for routine sterol analysis in EVOO.