Exploratory analysis of human urine by LC–ESI-TOF MS after high intake of olive oil: understanding the metabolism of polyphenols

Abstract

Olive oil polyphenols have important biological properties which closely depend on their bioavailability, therefore it is essential to understand how polyphenols are absorbed, metabolized and eliminated from the body. An analytical methodology based on rapid resolution liquid chromatography (RRLC) coupled to mass spectrometry detection with a time of flight analyzer (RRLC-ESI-TOF MS) was developed for the determination of the main olive oil phenolic compounds and their metabolites in human urine. Urine samples from ten healthy volunteers were collected before and 2, 4 and 6 h after the intake of 50 mL of extra-virgin olive oil. The proposed method includes liquid-liquid extraction with ethyl acetate that provides extraction recoveries of the phenolic compounds studied between 35 and 75% from spiked urine samples. Good repeatability was obtained, since the relative standard deviations (RSDs) of peak areas in the intra- and inter-day studies were 4.3 and 6.5%, respectively. Statistical studies allowed us to discriminate between the urine samples before and after the intake, and facilitated to find out the m/z values responsible of this discrimination. Based on the very accurate mass information and the isotopic pattern provided by TOF-MS analyzer, together with other available information, ten of these biomarkers and more than 50 metabolites, obtained through phase I and phase II biotransformation reactions, were tentatively identified. Additionally, kinetic studies of the metabolites identified as possible biomarkers were developed, obtaining maximal values in the first two hours for most compounds.