Separation and determination of sterols in olive oil by HPLC-MS

This study presents the first liquid chromatographic method for the identification and quantification of seven phytosterols in olive oil. Sterols were identified and quantified by liquid chromatography with mass spectrometry detection in positive APCI (atmospheric pressure chemical ionisation) mode. The samples were saponified by refluxing with 2 N ethanolic KOH, and the non-saponificable fraction was extracted with diethyl ether. This fraction was subjected to thin layer chromatography (TLC) on silica gel plates and then the band of sterols was isolated and extracted with methanol. Sterols were quantified by LC-MS, on a Waters Atlantis 5 μm dC₁₈2,1 × 150 mm column with a gradient of acetonitrile/water (0.01% acetic acid) at a flow of 0.5 mL min⁻¹; column temperature 30 °C. The method presents values between 123 and 677 ng mL⁻¹ for detection limits, with relative standard deviations between 4.0% and 5.4% at a concentration of 5 mg L⁻¹ for each sterol. Sterol contents were determined in a virgin olive oil, a refined olive oil, an olive-pomace oil and a crude olive-pomace oil.