Hydrogen/Deuterium Exchange Mass Spectrometry for Probing the Isomeric Forms of Oleocanthal and Oleacin in Extra Virgin Olive Oils.

Reversed-phase liquid chromatography and electrospray ionization with Fourier-transform single and tandem mass spectrometry (RPLC-ESI-FTMS and FTMS/MS) were employed for the structural characterization of oleocanthal (OLEO) and oleacin (OLEA), two of the most important bioactive secoiridoids occurring in extra virgin olive oils (EVOOs). The existence of several isoforms of OLEO and OLEA was inferred from the chromatographic separation, accompanied, in the case of OLEA, by minor peaks due to oxidized OLEO recognized as oleocanthalic acid isoforms. The detailed analysis of the product ion tandem MS spectra of deprotonated molecules ([M-H]-) was unable to clarify the correlation between chromatographic peaks and specific OLEO/OLEA isoforms, including two types of predominant dialdehydic compounds, named Open Forms II, containing a double bond between carbon atoms C8 and C10, and a group of diasteroisomeric closed-structure (i.e., cyclic) isoforms, named Closed Forms I. This issue was addressed by H/D exchange (HDX) experiments on labile H atoms of OLEO and OLEA isoforms, performed using deuterated water as a co-solvent in the mobile phase. HDX unveiled the presence of stable di-enolic tautomers, in turn providing key evidence for the occurrence, as prevailing isoforms, of Open Forms II of OLEO and OLEA, different from those usually considered so far as the main isoforms of both secoiridoids (having a C=C bond between C8 and C9). It is expected that the new structural details inferred for the prevailing isoforms of OLEO and OLEA will help in understanding the remarkable bioactivity exhibited by the two compounds.

Bioactive Secoiridoids in Italian Extra-Virgin Olive Oils: Impact of Olive Plant Cultivars, Cultivation Regions and Processing.

In the last two decades, phenolic compounds occurring in olive oils known as secoiridoids have attracted a great interest for their bioactivity. Four major olive oil secoiridoids, i.e., oleuropein and ligstroside aglycones, oleacin and oleocanthal, were previously characterized in our laboratory using reversed-phase liquid chromatography with electrospray ionization-Fourier transform-mass spectrometry (RPLC-ESI-FTMS). The same analytical approach, followed by multivariate statistical analysis (i.e., Principal Component Analysis), was applied here to a set of 60 Italian extra-virgin olive oils (EVOO). The aim was to assess the secoiridoid contents as a function of olive cultivars, place of cultivation (i.e., different Italian regions) and olive oil processing, in particular two- vs. three-phase horizontal centrifugation. As expected, higher secoiridoid contents were generally found in olive oils produced by two-phase horizontal centrifugation. Moreover, some region/cultivar-related trends were evidenced, as oleuropein and ligstroside aglycones prevailed in olive oils produced in Apulia (Southern Italy), whereas the contents of oleacin and oleocanthal were relatively higher in EVOO produced in Central Italy (Tuscany, Lazio and Umbria). A lower content of all the four secoiridoids was generally found in EVOO produced in Sicily (Southern Italy) due to the intrinsic low abundance of these bioactive compounds in cultivars typical of that region.

Characterization of bioactive and nutraceutical compounds occurring in olive oil processing wastes.

RATIONALE: Several bioactive compounds, including phenolic acids and secoiridoids, are transferred from olive drupes to olive oil during the first stage of production. Here, the characterization of these low molecular weight (LMW) compounds in olive oil and in closely related processing materials, like olive leaves (OL) and olive mill wastewaters (OMW), was faced up, for the first time, by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF MS). METHODS: A novel binary matrix composed of 1,8-bis(tetramethylguanidino)naphthalene (TMGN) and 9-aminoacridine (9AA) (1:1 molar ratio), displaying excellent ionization properties at low levels of laser energy, was employed in reflectron negative ion mode by a MALDI TOF/TOF system equipped with a neodymium-doped yttrium lithium fluoride (Nd:YLF) laser (345 nm). MS/MS experiments were performed by using ambient air as the collision gas. RESULTS: Four major secoiridoids typically present in olive oil, i.e., the aglycones of oleuropein and ligstroside, and oleacein and olecanthal at m/z 377.1, 361.1, 319.1 and 303.1, respectively, were detected in virgin olive oil (VOO) extracts, along with some of their chemical/enzymatic hydrolysis by-products, such as elenolic (m/z 241.1), decarboxymethyl-elenolic acids (m/z 183.1) and hydroxytyrosol (m/z 153.1). Besides oleuropein aglycone and oleacein, hydroxylated derivatives of decarboxymethyl-elenolic acid and hydroxytyrosol were evidenced in OMW. CONCLUSIONS: While oleuropein was confirmed in OL extracts, several interesting phenolic compounds, including hydroxytyrosol, were recognized in OMW. The proposed approach based on the use of a novel binary matrix for MALDI MS/MS analyses of LMW bioactive compounds can be considered a promising analytical tool for a rapid screening of the phenolic fraction in olive oils and related processing wastes.

Tandem mass spectrometry characterization of a conjugate between oleuropein and hydrated cis-diammineplatinum(II).

RATIONALE: Oleuropein (Ole) has been claimed to mitigate cisplatin (CP)-induced acute injury in kidney and liver of mice. In vitro reactivity of hydrated CP species with Ole, and an Ole metabolite, hydroxytyrosol (HT), is of great interest as the preliminary step for gathering in vivo information on the possible physiological role of the Ole/HT-cis-diammineplatinum(II) (Ole/HT-cis-DAP) conjugate. METHODS: Reversed-phase liquid chromatography coupled to electrospray ionization mass spectrometry using a linear ion trap instrument (RPLC/ESI-MS) and tandem mass (MS/MS) measurements, both in positive and negative ion mode, revealed the molecular identity of platinum-based conjugates. RESULTS: The Ole-cis-DAP conjugate (i.e., C25 H36 N2 O13 PtII ) features two cis-ammine non-leaving ligands and a bidentate catechol ligand moiety belonging to Ole; the coordination of the central Pt(II) is square-planar with non-equivalent bond angles compared with the ideal arrangement of 90°. HT, the free Ole metabolite excreted in human urine, acts as bidentate O,O-donor ligand of cis-DAP as well. CONCLUSIONS: The first evidence, together with structural information, is provided about the in vitro formation of a conjugate between cis-DAP and Ole or its urinary metabolite HT. Presuming that such conjugates are also generated in vivo, the mechanisms by which they might contribute to reduce CP toxicity remain to be elucidated.

Exploring the Isomeric Precursors of Olive Oil Major Secoiridoids: An Insight into Olive Leaves and Drupes by Liquid-Chromatography and Fourier-Transform Tandem Mass Spectrometry.

Secoiridoids play a key role in determining health benefits related to a regular consumption of extra-virgin olive oil (EVOO), in which they are generated from precursors of the same class naturally occurring in drupes and leaves of the olive (Olea europaea L.) plant. Here, reversed-phase liquid chromatography coupled to electrospray ionization and Fourier-transform single/tandem mass spectrometry (RPLC-ESI-FTMS and MS/MS) was employed for a structural elucidation of those precursors. The presence of three isoforms in both matrices was assessed for oleuropein ([M-H]- ion with m/z 539.1770) and was emphasized, for the first time, also for ligstroside (m/z 523.1821) and for the demethylated counterparts of the two compounds (m/z 525.1614 and 509.1665, respectively). However, only the prevailing isoform included an exocyclic double bond between carbon atoms C8 and C9, typical of oleuropein and ligstroside; the remaining, less abundant, isoforms included a C=C bond between C8 and C10. The same structural difference was also observed between secoiridoids named elenolic acid glucoside and secoxyloganin (m/z 403.1246). This study strengthens the hypothesis that secoiridoids including a C8=C10 bond, recently recognized as relevant species in EVOO extracts, arise mainly from specific enzymatic/chemical transformations occurring on major oleuropein/ligstroside-like precursors during EVOO production, rather than from precursors having that structural feature.

Bioactive Compounds in Waste By-Products from Olive Oil Production: Applications and Structural Characterization by Mass Spectrometry Techniques.

In recent years, a remarkable increase in olive oil consumption has occurred worldwide, favoured by its organoleptic properties and the growing awareness of its health benefits. Currently, olive oil production represents an important economic income for Mediterranean countries, where roughly 98% of the world production is located. Both the cultivation of olive trees and the production of industrial and table olive oil generate huge amounts of solid wastes and dark liquid effluents, including olive leaves and pomace and olive oil mill wastewaters. Besides representing an economic problem for producers, these by-products also pose serious environmental concerns, thus their partial reuse, like that of all agronomical production residues, represents a goal to pursue. This aspect is particularly important since the cited by-products are rich in bioactive compounds, which, once extracted, may represent ingredients with remarkable added value for food, cosmetic and nutraceutical industries. Indeed, they contain considerable amounts of valuable organic acids, carbohydrates, proteins, fibers, and above all, phenolic compounds, that are variably distributed among the different wastes, depending on the employed production process of olive oils and table olives and agronomical practices. Yet, extraction and recovery of bioactive components from selected by-products constitute a critical issue for their rational valorization and detailed identification and quantification are mandatory. The most used analytical methods adopted to identify and quantify bioactive compounds in olive oil by-products are based on the coupling between gas- (GC) or liquid chromatography (LC) and mass spectrometry (MS), with MS being the most useful and successful detection tool for providing structural information. Without derivatization, LC-MS with electrospray (ESI) or atmospheric pressure chemical (APCI) ionization sources has become one of the most relevant and versatile instrumental platforms for identifying phenolic bioactive compounds. In this review, the major LC-MS accomplishments reported in the literature over the last two decades to investigate olive oil processing by-products, specifically olive leaves and pomace and olive oil mill wastewaters, are described, focusing on phenolics and related compounds.

Influence of Horizontal Centrifugation Processes on the Content of Phenolic Secoiridoids and Their Oxidized Derivatives in Commercial Olive Oils: An Insight by Liquid Chromatography-High-Resolution Mass Spectrometry and Chemometrics.

Reversed-phase liquid chromatography with electrospray ionization-high-resolution/accuracy Fourier transform mass spectrometry (RPC-ESI-FTMS) and chemometrics were exploited to evaluate the influence of horizontal centrifugation by two- or three-phase decanters on the content of major phenolic secoiridoids in extravirgin olive oils (EVOOs). Despite the occurrence of other potential sources of variability typical of commercial olive oils, horizontal centrifugation was found to play a primary role, with a general increase of secoiridoid content occurring when two-phase decanters were used. As emphasized by principal component analysis (PCA), the increase involved preferentially oleacin and oleocanthal, when oxidative deterioration was purposely minimized during and/or after production, and oleuropein and ligstroside aglycones, when no vertical centrifugation was performed at the end of the productive cycle. The influence of the type of horizontal centrifugation was also emphasized by the elaboration of RPC-ESI-FTMS data based on hierarchical cluster analysis (HCA) and linear discriminant analysis (LDA).

A comprehensive study of oleuropein aglycone isomers in olive oil by enzymatic/chemical processes and liquid chromatography-Fourier transform mass spectrometry integrated by H/D exchange.

A comprehensive structural characterization of the complex family of isomeric forms related to Oleuropein aglycone (OA) detected in virgin olive oil (VOO) was performed by reverse phase liquid chromatography with electrospray ionization and Fourier-transform mass spectrometry (RPLC-ESI-FTMS), integrated by enzymatic/chemical reactions performed on Oleuropein, the natural precursor of OA. First, some of the OA-related isomers typically observed in VOO extracts were generated upon enzymatic hydrolysis of the glycosidic linkage of Oleuropein. This step mimicked the process occurring during olive drupes crushing in the first stage of oil production. The incubation of the enzymatic reaction mixture at a more acidic pH was subsequently performed, to simulate the conditions of olive paste malaxation during oil production. As a result, further isomeric forms were generated and the complex chromatographic profile typically observed for OA in olive oil extracts, including at least 13 different peaks/bands/groups of peaks, was carefully reproduced. Each of those chromatographic features could be subsequently assigned to specific types of OA-related isomers, belonging to one of four structurally different classes. Specifically, diastereoisomers/geometrical isomers corresponding to two different types of open-structure forms and to as many types of closed-structure, di-hydropyranic forms of OA, characterized by the presence of one or two carbonyl groups, according to the case, were evidenced. In addition, the presence of stable enolic/dienolic tautomers, providing an indirect structural confirmation for some OA isomers, was ascertained through RPLC-ESI-FTMS analyses performed under H/D exchange conditions, i.e. in the presence of deuterated water as one of the mobile phase solvents.

Structural characterization of the ligstroside aglycone isoforms in virgin olive oils by liquid chromatography-high-resolution Fourier-transform mass spectrometry and H/Dexchange.

A systematic structural characterization of the isomeric forms related to ligstroside aglycone (LA), one of the most relevant secoiridoids contained in virgin olive oils, was performed using reverse phase liquid chromatography with electrospray ionization Fourier-transform single and tandem mass spectrometry, operated in negative ion mode (RPLC-ESI(-)-FTMS and FTMS/MS). The high mass resolution and accuracy provided by the adopted orbital trap mass analyzer enabled the recognition of more than 10 different isomeric forms of LA in virgin olive oil extracts. They were related to four different types of molecular structure, two of which including a dihydropyranic ring bearing one or two aldehydic groups, whereas the others corresponded to dialdehydic open-structure forms, differing just for the position of a C═C bond. The contemporary presence of enolic or dienolic tautomers associated to most of these compounds, stable at room temperature (23°C), was also assessed through RPLC-ESI-FTMS analyses operated under H/D exchange conditions, ie, by using D2 O instead of H2 O as co-solvent of acetonitrile in the RPLC mobile phase. As discussed in the paper, the results obtained for LA indicated a remarkable structural similarity with oleuropein aglycone (OA), the most abundant secoiridoid of olive oil, whose isoforms had been previously characterized using the same analytical approach.

Reactivity of 4-thiothymidine with Fenton reagent investigated by UV-visible spectroscopy and electrospray ionization mass spectrometry.

The reactivity of the sulfur-containing nucleoside 4-thio-(2'-deoxy)-thymidine usually abbreviated as 4-thio-thymidine, (S4 -TdR) under Fenton conditions, ie, in the presence of H2 O2 and catalytic amounts of Fe(II), was investigated by UV-vis spectroscopy and electrospray ionization single and tandem mass spectrometry (ESI-MS and MS/MS). S4 -TdR hydroxylated on the S atom was found to be a key reaction intermediate, ultimately leading to (2'-deoxy)-thymidine usually abbreviated as thymidine, (TdR) as the main reaction product. This finding was in accordance with the outcome of the reaction between S4 -TdR and H2 O2 , previously investigated in our laboratory. On the other hand, the additional presence of •OH radicals, induced by the Fe(II)/H2 O2 combination, led to the increased generation of another interesting S4 -TdR product, already observed after its reaction with H2 O2 alone, ie, the covalent dimer including a SS bridge between two S4 -TdR molecules. More importantly, multihydroxylated derivatives of S4 -TdR and TdR were detected as peculiar products obtained under Fenton conditions. Among them, a product bearing an OH group both on the methyl group linked to the thymine ring and on the C5 atom of the ring was found to prevail. The results obtained during this study, integrated by those found previously in our laboratory, indicate 4-thiothymidine as a promising molecular probe for the recognition, through a careful characterization of its reaction products, of the prevailing species among reactive oxygen species (ROS) corresponding to singlet-state oxygen, hydrogen peroxide, and hydroxylic radical.