UHPLC-MS Analysis and Quantification of Ferulic Acid after Digestion

BSG extract freeze-dried digested samples (1 mg) was solubilized in 1 mL of water and treated with 3 mL of ice-cold acetonitrile. The sample was centrifuged for 15 min at 16,000 rpm (Hermle®, Hamburg, Germany), and the solvent was evaporated with nitrogen; the supernatant was solubilized in 50:50 methanol/water 0.1% formic acid and analyzed.

HUPLC-MS/MS analyses for the identification and quantification of ferulic acid in the digested sample were performed on a Dionex Ultimate 3000 UHPLC system (Thermo Fisher Scientific, Milan, Italy) consisting of two pumps, an autosampler, and a column oven. The system was coupled to a Linear Ion Trap Mass Spectrometer LTQ XL (Thermo Fisher Scientific, Milan, Italy) equipped with an electrospray source (ESI).

The separation was performed on a LUNA® OMEGA POLAR C18 column (L × ID) 150 mm × 2.1 mm, 3 μm (Phenomenex, Milan, Italy). The mobile phases used were A) 0.1% HCOOH in H2O and B) ACN, with the following gradient: 0–5 min, 2% B; 5–15 min, 2–18% B; 15–30 min 18–95% B and hold for 5 min; returning to initial conditions in 0.2 min. The flow rate was set to 0.3 mL/min. Column oven was set to 45 ◦C, and 5 μL of extract were injected.

The ESI source was operated in the negative mode. MS/MS analyses were conducted in full mode, using the following ion with m/z 195.2 corresponding to ferulic acid, collision energy: 35.0 V, and scan range m/z 50–200. To optimize the MS operating conditions, a preliminary experiment was performed: 10 μg/mL ferulic acid (H2O/MeOH: 50/50 with 0.1% formic acid) solutions were directly infused in the ESI interface at a flow rate of 25 μL/min into the mass spectrometer. Optimized conditions were as follows: sheath gas 60, capillary temperature 220 ◦C, auxiliary gas 25, spray voltage 4.5 kV, and capillary voltage −26.13 V for negative ionization mode. Ferulic acid (Sigma Aldrich, Milan, Italy) was selected as the external standard for the quantitation. Stock solution (1 mg/mL) was prepared in H2O/MeOH: 50:50 with 0.1% formic acid, and the calibration curve (y = 3669.9x − 50.911) was obtained in the concentration range of 0.62–1 μg/mL (R2 = 0.9973).
