Compounds were separated using a Zorbax SB-C18 reversed-phase analytical column (100 mm × 3.0 mm i.d., 5 μm particle) fitted with a guard column Zorbax SB-C18, both operated at 40°C. Sterols were separated under isocratic conditions using a mobile phase consisting of 10:90 (v/v) methanol and acetonitrile. The flow rate was 1 mL/min and the injection volume was 5 μL. Mass spectrometry analysis was performed on an Agilent Ion Trap 1100 VL mass spectrometer with atmospheric pressure chemical ionization (APCI) interface. The instrument was operated in positive ion mode. Operating conditions were optimized in order to achieve maximum sensitivity values: gas temperature (nitrogen) 325°C at a flow rate of 7 L/min, nebulizer pressure 60 psi and capillary voltage -4000 V.
The identification of sterols was performed by comparing the retention times and mass spectra with those of standards in the same chromatographic conditions. To avoid or limit the interference from background, the multiple reactions monitoring analysis mode was used instead of single ion monitoring (e.g., MS/MS instead of MS). Linearity of calibration curves was very good (R2 > 0.998), with detection limits in the range of 69 to 3312 ng/mL for ergosterol, 62 to 2952 ng/mL for brassicasterol, 59 to 2808 ng/mL for campesterol, 136 to 6528 ng/mL for stigmasterol, and 132 to 6336 ng/mL for β-sitosterol. The results are expressed as μg per mL of extract (μg/mL).
The software ChemStation (vA09.03) and DataAnalysis (v5.3) from Agilent, United States were used for the acquisition and analysis of chromatographic data (Vlase et al., 2013 (link)).
The identification of sterols was performed by comparing the retention times and mass spectra with those of standards in the same chromatographic conditions. To avoid or limit the interference from background, the multiple reactions monitoring analysis mode was used instead of single ion monitoring (e.g., MS/MS instead of MS). Linearity of calibration curves was very good (R2 > 0.998), with detection limits in the range of 69 to 3312 ng/mL for ergosterol, 62 to 2952 ng/mL for brassicasterol, 59 to 2808 ng/mL for campesterol, 136 to 6528 ng/mL for stigmasterol, and 132 to 6336 ng/mL for β-sitosterol. The results are expressed as μg per mL of extract (μg/mL).
The software ChemStation (vA09.03) and DataAnalysis (v5.3) from Agilent, United States were used for the acquisition and analysis of chromatographic data (Vlase et al., 2013 (link)).
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