1576 Original Papers From Olive Drupes to Olive Oil. An HPLC-Orbitrap-based Qualitative and Quantitative Exploration of Olive Key Metabolites Authors Periklis Kanakis1, Aikaterini Termentzi1, Thomas Michel1, Evagelos Gikas2, Maria Halabalaki 1, Alexios-Leandros Skaltsounis1 Affiliations 1 Laboratory of Pharmacognosy and Natural Products Chemistry, School of Pharmacy, University of Athens, Athens, Greece 2 Laboratory of Pharmaceutical Chemistry, School of Pharmacy, University of Athens, Athens, Greece Key words Abstract identified based on HRMS and HRMS/MS data. l" olive oil production ! Nine biologically and chemically significant me- l" olive drupes The aim of the current study was the qualitative tabolites were quantitatively determined l" ‑ ‑ HPLC Orbitrap HRMS/MS exploration and quantitative monitoring of key throughout the four production steps. Drupes l" oleocanthal olive secondary metabolites in different pro- and paste were found to be rich in several compo- l" oleacein duction steps (drupes, paste, first and final oil) nents, which are not present in the final oil. The throughout a virgin olive oil production line. The current study discloses the chemical nature of dif- Greek variety Koroneiki was selected as one of the ferent olive materials in a successive and inte- most representative olives, which is rich in bio- grated way and reveals new sources of high added logical active compounds. For the first time, an value constituents of olives. HPLC-Orbitrap platform was employed for both qualitative and quantitative purposes. Fifty-two Supporting information available online at components belonging to phenyl alcohols, secoir- http://www.thieme-connect.de/ejournals/toc/ idoids, flavonoids, triterpenes, and lactones were plantamedica Introduction thal. These compounds show antimicrobilal, anti- ! inflammatory, and hypoglycaemic effects and are In 2011, the European Food Safety Authority (EF- considered responsible for the antioxidant prop- SA) published a scientific opinion suggesting that erties of VOO [3–5]. Apart from phenols and se- virgin olive oil (VOO) could be considered as a coiridoids, triterpenes, such as maslinic and olea- Downloaded by: Ufficio Servizi Bibliotecnici. Copyrighted material. received April 29, 2013 beneficial health agent based on the numerous nolic acid, are also characteristic secondary me- revised July 1, 2013 scientific publications that underline its biologi- tabolites, abundant in VOO, and contribute to sev- accepted August 11, 2013 cal impact. Most of those studies have shown as- eral of its biological effects. l" Fig. 1 and Fig. 1S of Bibliography sociations between a VOO-rich diet and a de- the supplementary data illustrate representative DOI http://dx.doi.org/ creased cardiovascular risk, stroke incidences, secondary metabolites found in VOO with a docu- 10.1055/s-0033-1350823 and type 2 diabetes. Specifically, VOO has been mented biological impact. Published online September 26, 2013 shown to possess antiatherosclerotic effects and Despite the high significance of the olive polyphe- Planta Med 2013; 79: its consumption has been shown to decrease nols, their final concentration in the oil is really 1576–1587 © Georg Thieme plasma low-density lipoprotein (LDL) and choles- questionable, since the process during oil pro- Verlag KG Stuttgart · New York · terol levels [1]. In other studies, VOO showed po- duction can destroy, degrade, or simply remove ISSN 0032‑0943 tent antihaemostatic and anti-inflammatory to waste, large quantities of these valuable sec- Correspondence properties, as well as an improved effect on endo- ondary metabolites. The production of oil is more Prof. Alexios-Leandros thelial function and insulin sensitivity [2]. One of dynamic than a simple extraction procedure, Skaltsounis Laboratory of Pharmacognosy the most important groups of bioactive secondary since important enzymatic functions are acti- and Natural Products Chemistry metabolites in VOO is phenolic alcohols, e.g., tyro- vated transforming initial bioactive molecules School of Pharmacy sol and hydroxytyrosol. These alcohols form es- found in drupes to other derivatives, finally con- University of Athens Panepistimioupoli Zografou ters with elenolic acid derivatives to give glycosy- sumed in oil. Furthermore, the comprehension of 15771 Athens lated secoiridoids, such as oleuropein and ligstro- the variability of crucial olive secondary metabo- Greece side, the corresponding aglycons and decarboxy- lites throughout the olive oil production proce- Phone: + 302107274598 Fax: + 302 0107274594 methyl forms with different configurations of the dure is of great importance. The synthesis of sev- [email protected] elenolic acid moiety, e.g., oleacein and oleocan- eral of these valuable components is often de- Kanakis P et al. From Olive Drupes… Planta Med 2013; 79: 1576–1587 Original Papers 1577 Fig. 1 Representative structures of olive secondary metabolites that have been quantified throughout the production steps of VOO (the numbering of compounds is in accordance with l" Table 1). manding, time consuming, and expensive, e.g., oleocanthal [6,7]. Additionally, in a modern consuming market, compounds of nat- ural origin, rather than synthetic (organic, natural materials for food supplements and cosmetics), are requested. Thus, it is crit- Fig. 2 Graphical illustration of the basic production steps of a two-phase ical to explore the entire procedure of olive oil production quali- olive oil mill. The different materials under investigation are highlighted as tatively and quantitatively, and define the chemical nature of well as the quantitative alterations of some biologically important second- each intermediate material. Along these lines, the determination ary metabolites. MFOA: monoaldehydic form of oleuropein aglycon; MFLA: monoaldehydic form of ligstroside aglycon. of new or optimum sources of key bioactives of olives is of great (Color figure available online only.) important for the isolation thereof or the creation of enriched ex- tracts. Up to now, studies are characterised by the lack of a holistic, com- plete, and successive approach that could investigate the entire Thus, the aim of the present study was the qualitative and quan- metabolic content of different materials of the olive oil pro- titative monitoring of important olive secondary metabolites, duction process. Most of the studies concern determination of throughout the whole olive oil production procedure, incorpo- Downloaded by: Ufficio Servizi Bibliotecnici. Copyrighted material. certain phenols in specific samples, such as single paste, drupes, rating an HPLC‑LTQ-Orbitrap platform. The basic steps of the or oil extracts. Consequently, this fragmented approach allows olive oil production procedure, from the drupes to final oil neither the comprehensive investigation of olive constituents (drupes, paste, first oil, final oil, waste), at a commercial two- nor their biotransformation, and therefore the quality of olive oil. phase oil mill are defined and the most important Greek variety Regarding the analytical methods used for the quantitative deter- Koroneiki, at the late ripening stage, was investigated. A detailed minations of phenols and/or secoiridoids, most of the studies HPLC-HRMS and HRMS/MS-based study for the complete charac- have been performed on the basis of the UV‑Vis absorption [8– terisation of the chemical constituents of each production step 10]. There are a limited number of studies utilising LC‑MS plat- was performed resulting in the identification of more than 50 forms for quantitative analysis of components in specific olive secondary metabolites, with high confidence. Furthermore, given samples. Regarding the analysers, mostly triple quadrupoles the fact that the published data are controversial, quantitative (TQ) have been used [11,12], while recently some studies incor- monitoring of nine main components of olives with high biologi- porating TOF technology have been published [13]. cal importance (hydroxytyrosol, tyrosol, oleacein, oleocanthal, HRMS analysers such as TOF and Orbitrap have become competi- oleuropein, ligstroside, monoaldehydic form of oleuropein agly- tive tools for the characterisation of complicated components in con – MFOA, monoaldehydic form of ligstroside aglycon – MFLA, complex matrices. The latest generations of HRMS instruments, and maslinic acid) was also carried out. such as Orbitrap platforms, show improved resolution and stabil- ity of accurate mass measurements, and at the same time great quantitative potential [14]. Given these advantages, Orbitrap-MS quantitative determination is an alluring platform for confident and rapid quantitative and, simultaneously, qualitative monitor- ing of small molecules in complex mixtures. Kanakis P et al. From Olive Drupes… Planta Med 2013; 79: 1576–1587 1578 Original Papers Fig. 3 Comparative base peak chromatograms of all the extracts from each step of the olive oil pro- duction procedure. Representative secondary me- tabolites are annotated. (Color figure available online only.) Results and Discussion HPLC‑MS/MS systems for the quantitative determination of sev- ! eral major olive phenolics, such as hydroxytyrosol, tyrosol, oleur- Even if olive oil is a widely investigated product due to its nutri- opein, and others [12,22]. However, the application of HPLC‑LTQ- tional and therapeutic values, the studies regarding the constitu- Orbitrap is something that has never been applied before. Specif- ents of olive drupes as well as the intermediate materials