Alliance 2695 lc system

Analyses of phenolic compounds were performed on an Alliance 2695LC system (Waters) equipped with a 2998 diode array detector (DAD) set at 220–540 nm detection range, and a Symmetry C18 reverse phase column (100 × 4.6mm) (Waters). The eluent was a mixture of solvent A (water acidified with 0.1% formic acid) and solvent B (acetonitrile grade HPLC). A 30 μl volume of the phenolic extract was filtered through a 0.4 μm pore diameter syringe filter prior injection. Elution gradient was 5%–40% (B) from 0 to 18 min, 40%–90% (B) from 18 to 20 min and 90% (B) from 20 to 24 min at a flow rate of 0.5 mL/min and 35 °C. Individual compound identification and quantification was obtained by comparison of retention times and using a calibration curve of pure standard.

Analyses were performed by an Alliance 2695 LC system (Waters, Milford, MA, USA) coupled with a triple-quadrupole tandem Quattro Micro mass spectrometer (Waters, Milford, MA, USA). Instrumental control is the Mass Lynx 4.1 software using for acquisition and processing of the data. The LC separation was performed on an Agilent Zorbax SB-C18 column (150 2.1 mm i.d. 5 mm, Agilent Technologies, Wilmington, DE, USA) by the mobile phase consisting of acetonitrile and deionized water (40:60, v/v) containing 10 mM at a flow rate of 0.8 mL/min with a security guard column (12.5 2.1 mm i.d. 5 mm, Agilent Zorbax SB-C18, DE, USA). The autosampler temperature was maintained at 15 °C. The total LC run time was 10 min with the column temperature kept at 30 °C.
The typical operating source condition for MS detector with an electrospray ionization (ESI) interface in negative ion mode. The detertion parameters were optimized as follows: capillary voltage, 2.7 kV; cone voltage, 45 V; source temperature, 110 °C; desolvation temperature, 350 °C; desolvation gas flow (nitrogen), 450 L/h; collision energy 28 V for limonin and 25 V for nimodipine; argon was used as the collision gas with the gas pressure of 3.0 × 10⁻³mbar.