VOO phenolics were isolated by SPE on a diol-bonded phase cartridge (Supelco, Bellefonte, PA) following a previously described procedure [23] (link). A solution of p-hydroxyphenyl-acetic acid (4.64×10−2 mg/mL) and o-coumaric acid (9.6×10−3 mg/mL) in methanol was used as internal standard in this extraction procedure. An aliquot (0.5 mL) of standard solution was added to each oil sample (2.5 g) before phenolic extraction. Two phenolic extracts were obtained from each VOO.
VOO phenolic extracts were further analyzed by HPLC in a Beckman Coulter liquid chromatographic system equipped with a System Gold 168 detector, a solvent module 126 and a Mediterranean Sea 18 column (4.0 mm i.d.×250 mm, particle size 5 μm) (Teknokroma, Barcelona, Spain) following a previously described methodology [24] (link). The quantification of phenols (except ferulic acid) and lignans was carried out at 280 nm using p-hydroxyphenyl-acetic acid as internal standard. The quantification of flavones and ferulic acid was done at 335 nm using o-coumaric acid as internal standard. The identification of compounds was confirmed by HPLC-MS using the same chromatographic system connected on-line with a MAT95 magnetic sector mass spectrometer (Finnigan Mat, Bremen, Germany) equipped with an ESI-II electrospray inonization (ESI) interface with the same column and gradient conditions. The ESI mass spectra in the positive mode were obtained under the following conditions: capillary temperature, 220°C; lens, skimmer, and octapole voltages were set to get optimal response for a pattern solution of reserpine. Nitrogen at 200 kPa was used as the sheath gas. Afterward, partial defocusing of interface was done in order to generate moderate collision-induced dissociation (CID) inside the ionic transport region. Under these conditions, the spectra show enough ionic fragmentation to verify structural information from the protonated molecular ion.
VOO phenolic extracts were further analyzed by HPLC in a Beckman Coulter liquid chromatographic system equipped with a System Gold 168 detector, a solvent module 126 and a Mediterranean Sea 18 column (4.0 mm i.d.×250 mm, particle size 5 μm) (Teknokroma, Barcelona, Spain) following a previously described methodology [24] (link). The quantification of phenols (except ferulic acid) and lignans was carried out at 280 nm using p-hydroxyphenyl-acetic acid as internal standard. The quantification of flavones and ferulic acid was done at 335 nm using o-coumaric acid as internal standard. The identification of compounds was confirmed by HPLC-MS using the same chromatographic system connected on-line with a MAT95 magnetic sector mass spectrometer (Finnigan Mat, Bremen, Germany) equipped with an ESI-II electrospray inonization (ESI) interface with the same column and gradient conditions. The ESI mass spectra in the positive mode were obtained under the following conditions: capillary temperature, 220°C; lens, skimmer, and octapole voltages were set to get optimal response for a pattern solution of reserpine. Nitrogen at 200 kPa was used as the sheath gas. Afterward, partial defocusing of interface was done in order to generate moderate collision-induced dissociation (CID) inside the ionic transport region. Under these conditions, the spectra show enough ionic fragmentation to verify structural information from the protonated molecular ion.
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