Determination of free and protein bound selenoaminoacids in regional olive oils

SABIER TORRES; FERNANDA SILVA; PABLO PACHECO

Simposio; 13th Rio Symposium on Atomic Spectrometry; 2014

Olive oils present low protein content and the presence of selenoaminoacids is unknown. For this reason, the expected selenoaminoacids concentration to be found in olive oil is scarce, but presumable essential and nutritionally important. This study reports a method to determine selenoaminoacids by extraction and preconcentration on an XAD resin followed by determination by reversed phase chromatography [RPC] coupled to inductively coupled plasma mass spectrometry [ICP MS]. Free selenoaminoacids, selenomethionine [Se-Met], selenomethylselenocysteine [Se-MetSeCys], and selenocysteine [Se-Cys]; were extracted from olive oils by a method involving olive oil mixing with hexane followed by water/methanol extraction (80:20) in ultrasonic bath. Methanol was eliminated and the extracts were loaded on an XAD 1080 resin, packed in a conical FI minicolumn. Elution was performed with a 0.5 M formic acid solution. Eluate was injected in a C18 column for RPC, employing as mobile phase 98% of a 0.1% (v v-1) trifluoroacetic acid solution and 2% methanol. The developed methods reached a detection limit of 0.09 µg kg-1, a precision of 10% (RSD, n = 6), and an enhancement factor of 60-fold (6 for the extraction system and 10 for the preconcentration approach). The only detected Se species in the olive oils was Se-MetSeCys in concentrations ranging from 2.0 to 8.3 µg kg-1 [1]. Total selenium determination was performed by ICP MS after microwave-assisted digestion and selenium concentrations in olive oil samples ranged from 62.8 ± 1.6 to 117.4 ± 3.0 µg kg-1. Determination of selenoaminoacids bound to proteins was carried as follows. Extraction was performed by addition of a cold acetone/hexane solution to olive oil and centrifugation at -2ºC. The precipitate containing proteins was solubilized with water:methanol (80:20) and introduced to size exclusion chromatography [SEC] coupled to ICP MS, monitoring Se and S. Fractions of 44-17 KDa containing Se and S associations, corresponding to proteins, were determined and collected. The proteins present in this fraction were hydrolyzed by acid microwave assisted digestion [MAD]. Hydrolisates were preconcentrated employing XAD 1080 minicolumn and analyzed by RPC-ICP MS. The selenoaminoacids concentration bound to proteins in the extracts was of 3.25 ± 1.0 µg kg-1. Se-Met, Se-MetSeCys and Se-Cys relative concentrations varied according to each olive oil sample analyzed, suggesting a possible relationship with their origin region.