To elucidate the elution profile
of the reference compounds, spiked AF extract was fractionated on
a reversed phase (RP) Phenomenex Kinetex C18 (100 mm × 2.1 mm,
5 μm pore size) column using an Agilent 1260 HPLC system equipped
with a binary pump, fraction collector, and diode array detector.
Data analysis and processing was performed with Agilent OpenLAB 1.1
Software. Spiked AF extract was injected (50 μL) at a flow rate
of 500 μL/min in 95% HPLC grade water and 5% MeOH. The solvent
gradient was held as such for 2 min and then increased linearly reaching
100% MeOH at 48 min. It was held at 100% MeOH for 2 min and then returned
to starting conditions over a period of 10 min to equilibrate the
column before the next injection. The column temperature was set at
35 °C. Two minute fractions were collected throughout the analytical
period, excluding the column equilibrium period, resulting in 25 fractions.
Each fraction was evaporated to dryness with a gentle stream of nitrogen
(at max 40 °C), the remaining residues were reconstituted in
300 μL of 5% MeOH and analyzed by LC–ESI–MS/MS
to determine the analyte recoveries.
of the reference compounds, spiked AF extract was fractionated on
a reversed phase (RP) Phenomenex Kinetex C18 (100 mm × 2.1 mm,
5 μm pore size) column using an Agilent 1260 HPLC system equipped
with a binary pump, fraction collector, and diode array detector.
Data analysis and processing was performed with Agilent OpenLAB 1.1
Software. Spiked AF extract was injected (50 μL) at a flow rate
of 500 μL/min in 95% HPLC grade water and 5% MeOH. The solvent
gradient was held as such for 2 min and then increased linearly reaching
100% MeOH at 48 min. It was held at 100% MeOH for 2 min and then returned
to starting conditions over a period of 10 min to equilibrate the
column before the next injection. The column temperature was set at
35 °C. Two minute fractions were collected throughout the analytical
period, excluding the column equilibrium period, resulting in 25 fractions.
Each fraction was evaporated to dryness with a gentle stream of nitrogen
(at max 40 °C), the remaining residues were reconstituted in
300 μL of 5% MeOH and analyzed by LC–ESI–MS/MS
to determine the analyte recoveries.