Copyright © 2009 John Wiley & Sons, Ltd. Biomed. Chromatogr. 2010; 24: 506–515 Research Article Received 12 June 2009, Accepted 21 July 2009 Published online in Wiley Interscience: 30 September 2009 (www.interscience.wiley.com) DOI 10.1002/bmc.1319 Simultaneous quantiication of oleuropein and its metabolites in rat plasma by liquid chromatography electrospray ionization tandem mass spectrometry Fotini N. Bazoti a,b , Evangelos Gikas b and Anthony Tsarbopoulos a,b * ABSTRACT: Oleuropein (OE) is the cardinal bioactive compound derived from Olea europaea and possesses numerous benei- cial properties for human health. However, despite the plethora of analytical methods that have studied the biological fate of olive oil-derived bioactive compounds, no validated methodology has been published to date for the simultaneous deter- mination of OE, along with all its major metabolites. In this study, a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI MS/MS) method has been developed and validated for the quantiication of OE, simultaneously with its main metabolites hydroxytyrosol, 2-(3,4-dihydroxyphenyl)acetic acid, 4-(2-hydroxyethyl)-2-methoxy-phenol or homovanillyl alcohol, 2-(4-hydroxy-3-methoxyphenyl)acetic acid or homovanillic acid, and elenolic acid in rat plasma matrix. Samples were analyzed by LC-ESI MS/MS prior to and after enzymatic treatment. A solid-phase extraction step with high mean recovery for all compounds was performed as sample pretreatment. Calibration curves were linear for all bioactive compounds over the range studied, while the method exhibited good accuracy, intra- and inter-day precision. The limit of detection was in the picogram range (per milliliterof plasma) for HT and OE and in the nanogram range (per milliliter of plasma) for the other analytes, and the method was simple and rapid. The developed methodology was successfully applied for the simultaneous quantiication of OE and its aforementioned metabolites in rat plasma samples, thus demonstrating its suitability for phar- macokinetics, as well as bioavailability and metabolism studies. Copyright © 2009 John Wiley & Sons, Ltd. Keywords: oleuropein; hydroxytyrosol; liquid chromatography; electrospray tandem mass spectrometry; plasma * Correspondence to: A. Tsarbopoulos, Department of Pharmacy, Laboratory of Instrumental and Pharmaceutical Analysis, University of Patras, Panepistimiopolis, Rio 265 04, Greece. E-mail: atsarbop@upatras.gr & atsarbop@gnhm.gr a Department of Pharmacy, Laboratory of Instrumental and Pharmaceutical Analysis, University of Patras, Panepistimiopolis, Rio 265 04, Greece b GAIA Research Center, Bioanalytical Department, The Goulandris Natural History Museum, 13 Levidou Street, Kiissia 145 62, Greece Abbreviations used: EA, elenolic acid; EVOO, extra virgin olive oil; HT, hydroxytyrosol; HTA, 2-(3,4-dihydroxyphenyl)acetic acid; HVA, homovanillic acid; HVAOH, homovanillyl alcohol; OE, oleuropein. Introduction Olea europea L. (Oleace) cultivation represents one of the most important and traditional agricultural activities in the Mediterranean countries and extra virgin olive oil (EVOO) is the major source of lipids (monounsaturated) in the Mediterranean diet. Olives and EVOO have been reported to contain phenolic compounds, like oleuropein (OE) and hydroxytyrosol (HT), which have been shown to possess antioxidant (Visioli and Galli, 2002; Manna et al., 2002; Briante et al., 2002), anti-inlammatory (Beauchamp et al., 2005; Martinez-Domingues, 2001), anti-ath- erogenic (Visioli and Galli, 2001; Carluccio et al., 2003) and anti- cancer (Owen et al., 2000; Trichopoulou et al. 1995) properties. They also possess antimicrobial activity against bacteria, yeasts, fungi and mycoplasma (Bisignano et al., 1999; Aziz et al., 1998; Furneri et al., 2002). Furthermore, these compounds ofer antivi- ral activity (Micol et al., 2005) and have been proven active against the infection and reproduction of HIV-1 virus (Lee-Huang et al., 2003); they have skin photoprotective properties (Saija and Uccella, 2001), while recent in vitro (Moosmann and Behl, 1999) and epidemiological (German and Walzem, 2000) studies have associated these compounds with protective properties against age-related diseases, like dementia (Howes et al., 2003; Bastianetto and Quirion, 2002), Alzheimer’s Disease (Hashimoto et al., 2004) and osteoporosis (Puel et al., 2004; Kanis, 1993). In vivo studies have shown that EVOO phenols are absorbed (Visioli et al., 2000; Mirò-Casas et al., 2003b) dose-dependently (Visioli et al., 2000) from the body and are excreted in urine (Caruso et al., 2001; Weinbrenner et al., 2004b; Marrugat et al., 2004), either in their free form or conjugated as glucuronides (Visioli et al., 2000), sulfate salts, methylated ethers and other derivatives (Tuck et al., 2002). The determination of the afore- mentioned compounds, as well as the study of their bioavail- ability (Coni et al., 2000) and metabolism (Caruso et al., 2001; D’Angelo et al., 2001) are of major importance for the evaluation of their biological role (Tuck and Hayball, 2002). The biological fate of olive oil-derived bioactive compounds (Tuck et al., 2001; Vissers et al., 2002) has been studied in plasma by HPLC-UV (Ruiz-Gutiérrez et al., 2000; Tsarbopoulos et al., 2003), 506