Determination of tyrosol and hydroxytyrosol by tandem spectrometry and isotope dilution approach in olive oil as marker of quality

This developed approach could be used for the assay of this molecules in biological fluids. Olive oil is the main source of fat in the Mediterranean diet; it is richer in polyphenols than refined olive oil or other unrefined oils and its consumption has been associated with a lower incidence of coronary heart disease and certain cancers. It's likely that the abundance of polyphenols in extra virgin olive oil, rather than the presence of monounsaturated fatty acids, is responsible for its well-known cardiovascular benefits. Phenols, which are strong radical scavengers and are responsible for the preservation and for the maintenance of the organoleptic properties of olive oil, reach a concentration up to 300 ppm; among those, hydroxytyrosol and tyrosol are two important compounds present in olive oils either as free or conjugated as secoiridoids d2-hydroxytyrosol and d2-tyrosol were synthesised according to literature. 100 mg of olive oil, was mixed with 10 μL of the 100 ppm solution of the internal standard and 900 μL of CH3OH. The resulting solution was stirred by vortex for 5 min and after was centrifuged at 2000 rpm for 3 min; the surnatant was then diluted (1/10) with water and injected in the instrument. The LC/MS analyses were carried out on a triple quadrupole LC 320 (Varian inc) equipped with an ESI source interfaced with an HPLC equipped with a C18 column, using a linear gradient H2O/CH3OH. The CID experiment of hydroxytyrosol displays few diagnostic fragments: the most abundant is the ion at m/z 123 that correspond to the formal loss of formaldehyde molecule; this hypothesis is confirmed by the analysis of the MS/MS spectrum of the labeled analogue which releases the neutral d2-formaldehyde forming an ion at the same m/z. The less abundant ion at m/z 122 present in the MS/MS spectra of either hydroxytyrosol and d2-hydroxytyrosol probably derives by the loss of an hydrogen atom to form the oxidized species quinone. A similar fragmentation pattern is present in the MS/MS spectra of tyrosol and d2-tyrosol: the main fragments, in fact, arises from the loss of formaldehyde or labeled formaldehyde and from the loss of a water molecule. Surprisingly, in the MS/MS spectrum of d2-tyrosol this signal is splitted in two peaks: that at m/z 120 and that at m/z 121; this is due, probably to a competitive 1-2 and 1-1 elimination; in particular the latter process involves an H/D isomerization. The LC-MS/MS analysis, under MRM condition, were carried out monitoring two diagnostic transitions for each analyte in order to assure the greatest specificity; more specifically, the assay has been performed by monitoring the most intense signal. In particular for the analyte hydroxytyrosol and d2-hydroxytyrosol were used the transitions leading to the loss of formaldehyde and d2- formaldehyde, respectively. For each analyte a calibration curve was obtained by injecting in triplicate standard solutions of analyte and internal standard. The accuracy test calculated preparing two spiked samples at known concentration, at two points which represent the upper and the lower limits of the calibration curve, was higher than 95% and the calculated values of LOQ and LOD corroborate the goodness of the proposed method.