2.5.2. Identification and Quantification of Phytosterols

The phytosterols in the HI extracts were determined according to an LC-UV-MS/MS method previously described [24]. In brief, apparatus and chromatographic analytical column were the same, but elution of compounds was performed in an isocratic mode, mobile phase containing acetonitrile/methanol (90:10, *v*/*v*), with a flow rate of 1 mL/min at 45 ◦C and 5 μL injection volume. For the detection of the analytes, the same ion trap mass spectrometer was used, fitted with an atmospheric pressure chemical ionization (APCI) interface in a positive mode. Operating conditions (dry nitrogen gas temperature 325 ◦C at a flow rate of 7 L/min, nebulizer pressure 60 psi, capillary voltage −4000 V) were adjusted for achieving maximum sensitivity values.

The full identification of compounds was performed by comparing the retention times and mass spectra with five external standards (ergosterol, brassicasterol, stigmasterol, campesterol, beta-sitosterol) (Table S3). Under the ionization conditions used for their determination, all sterols lose a water molecule and their pseudo-molecular ions are in the form M−H2O+H<sup>+</sup> [25]. The multiple reactions monitoring (MRM) analysis mode was used for detection in order to avoid or reduce the interference from the background. The quantification was performed based on the intensity of major daughter ions in the mass spectra [25]. The results were expressed as micrograms of phytosterols per dw involucre (μg/g dw).

The Agilent ChemStation (vA09.03) and DataAnalysis (v5.3) software were used for the acquisition and investigation of chromatographic data.
