3.4. Solid Phase Extraction (SPE)

An SPE method [24] previously used for the determination of amygdalin in almonds was adapted and optimized for our use. A Supelco Visiprep™ SPE vacuum manifold (Sigma-Aldrich, St. Louis, MO, USA) was used for this purpose. A vacuum of 10 in Hg (35 kPa) and a flowrate of about 1–2 drops/s was maintained throughout the process. A SPE cartridge (Sep-Pak Vac 3 cc (500 mg) C18 cartridge, Waters, Milford, MA, USA) was conditioned with 2 mL of methanol and equilibrated with 2 mL of water. 1 mL of the sample was loaded onto the column. An additional 1 mL of 0.1% FA in water was used to remove remaining residue in the extraction tube. The column was flushed with 2 mL of 0.1% FA in water. CNGs were finally eluted with 2 mL of varying proportions of methanol/water (0, 10, 20, 30 to 100% v/v). The extracts were dried under nitrogen gas, reconstituted into 0.1% FA in water and filtered through a 0.45 μm filter prior to UHPLC-MS/MS analysis.