Hydroxytyrosol (HT) from olive oil, a potent bioactive molecule with health benefits, has a poor bioavailability, its free form (free HT) being undetectable so far. This fact leads to the controversy whether attained HT concentrations after olive oil polyphenol ingestion are too low to explain the observed biological activities. Due to this, an analytical methodology to determine free HT in plasma is crucial for understanding HT biological activity. Plasma HT instability and low concentrations have been major limitations for its quantification in clinical studies. Here, we describe a method to detect and quantify free HT in human plasma by using liquid chromatography coupled to tandem mass spectrometry. The method encompasses different steps of sample preparation including plasma stabilization, protein precipitation, selective derivatization with benzylamine, and purification by solid-phase extraction. A high sensitivity (LOD, 0.3ng/mL), specificity and stability of HT is achieved following these procedures. The method was validated and its applicability was demonstrated by analyzing human plasma samples after olive oil intake. A pharmacokinetic comparison was performed measuring free HT plasma concentrations following the intake of 25mL of ordinary olive oil (nearly undetectable concentrations) versus an extra-virgin olive oil (Cmax=4.40ng/mL). To our knowledge, this is the first time that an analytical procedure for quantifying free HT in plasma after olive oil dietary doses has been reported. The present methodology opens the door to a better understanding of the relationship between HT plasma concentrations and its beneficial health effects.

This work was supported by grants from Instituto de Salud Carlos III FEDER, (PI14/00072), the CICYT-FEDER AGL2009-13517- C03-01 and AGL2012-40144-C03-01), grants from DIUE of the Generalitat de Catalunya (2014 SGR 680). CIBER de Fisiopatología de la Obesidad y Nutrición (CIBEROBN) is an initiative of the Instituto de Salud Carlos III, Madrid, Spain. J.R-M. was supported by a FI-DGR2012 predoctoral fellowship from the Generalitat de Catalunya. C.P-M. was supported by a Juan Rodés contract (JR15/00005).