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Walnut shells, often discarded as waste, hold hidden potential as a source of ellagitannins (ETs), compounds known for their promising antioxidant properties and health benefits. This study employed reversed-phase liquid chromatography (RPLC) coupled with Orbitrap-based high-resolution mass spectrometry (HRMS) via electrospray ionization (ESI) in negative polarity to investigate the ET profile in extracts of dried powdered walnut shells. Several compounds belonging to various ET families were successfully identified as deprotonated molecules ([M - H]-) and characterized, including mono-, di-, tri-, tetra-, and pentagalloyl glucopyranoses, as well as ETs containing the hexahydroxydiphenoyl (HHDP) group. Characteristic product ions were identified in HR tandem MS spectra and employed to recognize the ET landscape. Analysis revealed a complex picture with more than 10 isomers identified in some cases. However, the structural similarity and limitations in MS/MS data hindered the definitive identification of all isomers. Characterization of ETs featuring HHDP groups also remained challenging. Despite these restraints, the estimated total content of ETs suggests potential application in the food, pharmaceutical, and cosmetic industries of those extracts. These findings indicate that walnut shells can be considered a sustainable source of health-promoting compounds, contributing to a greener economy.

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Two protocols of protein extraction from Arthrospira platensis (spirulina) microalgae to study their proteome by mass spectrometry (MS) are here presented. The first is based on an aqueous buffer solution of Tris-HCl and the second on cold acetone. The identification of proteins was carried out by a bottom-up approach, which involves enzymatic digestion of extracted proteins followed by either matrix-assisted laser desorption ionization with time-of-flight (MALDI-TOF) MS or liquid chromatography (LC) coupled with electrospray ionization (ESI) and Fourier-transform tandem MS. While MALDI-TOF MS allowed for a fast peptide mass fingerprinting (PMF) check yet identifying less than 20 proteins in the extracted samples, the data-dependent acquisitions (DDA) mode of reversed-phase (RP) LC-ESI tandem FTMS/MS separations allowed us to recognize more than one hundred proteins by searching into dedicated spectral libraries. The application of MALDI-TOF MS analysis was found, however, of great support for preliminary investigations of cyanobacteria samples before proceeding with the RPLC-ESI-MS/MS DDA investigation, which definitively allows for a qualitative proteome analysis also of minor spirulina proteins in processed foodstuffs. Although the protein content in spirulina can be influenced by cultivation and environmental conditions, e.g., light intensity, climate, and water/air quality, here the qualitative chemical profiles of the examined samples were characterized by similar composition in high-quality proteins as phycocyanins and phycoerythrins.

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While edible algae might seem low in fat, the lipids they contain are crucial for good health and preventing chronic diseases. This study introduces a binary matrix to analyze all the polar lipids in both macroalgae (Wakame-Undaria pinnatifida, Dulse-Palmaria palmata, and Nori-Porphyra spp.) and microalgae (Spirulina-Arthrospira platensis, and Chlorella-Chlorella vulgaris) using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The key lies in a new dual matrix made by combining equimolar amounts of 1,5-diaminonaphthalene (DAN) and 9-aminoacridine (9AA). This combination solves the limitations of single matrices: 9AA is suitable for sulfur-containing lipids and acidic phospholipids, while DAN excels as an electron-transfer secondary reaction matrix for intact chlorophylls and their derivatives. By employing the equimolar binary matrix, a wider range of algal lipids, including free fatty acids, phospholipids, glycolipids, pigments, and even rare arsenosugarphospholipids were successfully detected, overcoming drawbacks related to ion suppression from readily ionizable lipids. The resulting mass spectra exhibited a good signal-to-noise ratio at a lower laser fluence and minimized background noise. This improvement stems from the binary matrix's ability to mitigate in-source decay effects, a phenomenon often encountered for certain matrices. Consequently, the data obtained are more reliable, facilitating a faster and more comprehensive exploration of algal lipidomes using high-throughput MALDI-MS/MS analysis.

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RATIONALE: 1,2-Diacyl-sn-glycero-3-phospho-O-[N-(2-hydroxyethyl)glycines] (PHEGs) are a class of rare aminophospholipids found specifically in brown algae, including kombu seaweed. Despite their potential importance in algal physiology, a comprehensive mass spectrometry (MS) characterization, useful to understand their biological behaviour, is still lacking. METHODS: To establish the structural regiochemical features of PHEGs, we employed hydrophilic interaction liquid chromatography (HILIC). Following separation, the isolated band of PHEGs was analyzed using MS techniques. This included multistage tandem MS experiments, performed in both positive and negative electrospray ionization modes at low and high resolution. RESULTS: By comparing MS/MS and MS3 spectra acquired in negative ion mode, the regiochemical rules for PHEG identification were established. The most abundant PHEG species in kombu seaweed, from both Laminaria ochroleuca (European Atlantic) and Laminaria longissima (Japan), was identified as PHEG 20:4/20:4. Less abundant species included PHEG 20:4/20:5 and hydroxylated forms of both PHEG 20:4/20:4 (i.e. 40:8;O) and 20:4/20:5 (40:9;O). The presence of a lyso PHEG 20:4 was consistently detected but at very low levels. CONCLUSIONS: This study employed MS analysis to elucidate the regiochemical patterns of PHEGs in kombu seaweed. We identified PHEG 20:4/20:4 as the dominant species, along with several less abundant variants, including hydroxylated forms. These findings provide valuable insights into the potential roles and metabolism of PHEGs in brown algae, paving the way for further investigation into their biological functions.

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Cyclic tetrapyrrole derivatives such as porphyrins, chlorins, corrins (compounds with a corrin core), and phthalocyanines are a family of molecules containing four pyrrole rings usually coordinating a metal ion (Mg, Cu, Fe, Zn, etc.). Here, we report the characterization of some representative cyclic tetrapyrrole derivatives by MALDI-ToF/ToF MS analyses, including heme b and c, phthalocyanines, and protoporphyrins after proper matrix selection. Both neutral and acidic matrices were evaluated to assess potential demetallation, adduct formation, and fragmentation. While chlorophylls exhibited magnesium demetallation in acidic matrices, cyclic tetrapyrroles with Fe, Zn, Co, Cu, or Ni remained steadfast against demetallation across all conditions. Phthalocyanines and protoporphyrins were also detectable without a matrix using laser desorption ionization (LDI); however, the incorporation of matrices achieved the highest ionization yield, enhanced sensitivity, and negligible fragmentation. Three standard proteins, i.e., myoglobin, hemoglobin, and cytochrome c, were analyzed either intact or enzymatically digested, yielding heme b and heme c ions along with accompanying peptides. Furthermore, we successfully detected and characterized heme b in real samples, including blood, bovine and cod liver, and mussel. As a result, MALDI MS/MS emerged as a powerful tool for straightforward cyclic tetrapyrrole identification, even in highly complex samples. Our work paves the way for a more comprehensive understanding of cyclic tetrapyrroles in biological and industrial settings, including the geochemical field, as these compounds are a source of significant geological and geochemical information in sediments and crude oils.

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This study investigates the ethanolic extract of dried walnut (Juglans regia L.) shells upon hammer milling (HM) and ball milling (BM) grinding processes. Marked differences were observed in the attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectra. The two extracts were investigated by reversed-phase liquid chromatography coupled with electrospray ionization and high-resolution mass spectrometry (RPLC-ESI-HRMS). Following enzymatic digestion, the fatty acids (FAs) were examined, and tandem MS of epoxidized species was applied to establish the C-C double bond position; the most abundant species were FA 18:2 Δ9,12, FA 18:1 Δ9, and FA 18:3 Δ9,12,15. However, no significant qualitative differences were observed between FAs in the two samples. Thus, the presence of potential active secondary metabolites was explored, and more than 30 phenolic compounds, including phenols, ellagic acid derivatives, and flavonoids, were found. Interestingly, the HM samples showed a high concentration of ellagitannins and hydrolyzable tannins, which were absent in the BM sample. These findings corroborate the greater phenolic content in the HM sample, as evaluated by the Folin-Ciocalteu test. Among the others, the occurrence of lanceoloside A at m/z 391.1037 [C19H20O9-H]-, and a closely related benzoyl derivate at m/z 405.1190 (C20H22O9-H]-), was ascertained. The study provides valuable information that highlights the significance of physical pre-treatments, such as mill grinding, in shaping the composition of extracts, with potential applications in the biorefinery or pharmaceutical industries.

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Food allergens are molecules, mainly proteins, that trigger immune responses in susceptible individuals upon consumption even when they would otherwise be harmless. Symptoms of a food allergy can range from mild to acute; this last effect is a severe and potentially life-threatening reaction. The European Union (EU) has identified 14 common food allergens, but new allergens are likely to emerge with constantly changing food habits. Mass spectrometry (MS) is a promising alternative to traditional antibody-based assays for quantifying multiple allergenic proteins in complex matrices with high sensitivity and selectivity. Here, the main allergenic proteins and the advantages and drawbacks of some MS acquisition protocols, such as multiple reaction monitoring (MRM) and data-dependent analysis (DDA) for identifying and quantifying common allergenic proteins in processed foodstuffs are summarized. Sections dedicated to novel foods like microalgae and insects as new sources of allergenic proteins are included, emphasizing the significance of establishing stable marker peptides and validated methods using database searches. The discussion involves the in-silico digestion of allergenic proteins, providing insights into their potential impact on immunogenicity. Finally, case studies focussing on microalgae highlight the value of MS as an effective analytical tool for ensuring regulatory compliance throughout the food control chain.

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Arsenic-containing lipids, also named arsenolipids (AsLs), are a group of organic compounds usually found in a variety of marine organisms such as fish, algae, shellfish, marine oils, and microorganisms. Numerous AsLs have been recognised so far, from simple compounds such as arsenic fatty acids (AsFAs), arsenic hydrocarbons (AsHCs), and trimethylarsenio fatty alcohols (TMAsFOHs) to more complex arsenic-containing species, of which arsenophospholipids (AsPLs) are a case in point. Mass spectrometry, both as inductively coupled plasma (ICP-MS) and liquid chromatography coupled by an electrospray source (LC-ESI-MS), was applied to organic arsenicals playing a key role in extending and refining the characterisation of arsenic-containing lipids in marine organisms. Herein, upon the introduction of a systematic notation for AsLs and a brief examination of their toxicity and biological role, the most relevant literature concerning the characterisation of AsLs in marine organisms, including edible ones, is reviewed. The use of both ICP-MS and ESI-MS coupled with reversed-phase liquid chromatography (RPLC) has brought significant advancements in the field. In the case of ESI-MS, the employment of negative polarity and tandem MS analyses has further enhanced these advancements. One notable development is the identification of the m/z 389.0 ion ([AsC10H19O9P]-) as a diagnostic product ion of AsPLs, which is obtained from the fragmentation of the deprotonated forms of AsPLs ([M - H]-). The pinpointing product ions offer the possibility of determining the identity and regiochemistry of AsPL side chains. Advanced MS-based analytical methods may contribute remarkably to the understanding of the chemical diversity characterising the metalloid As in natural organic compounds of marine organisms.

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Anacardic acids (AnAs) are important secondary metabolites that occur primarily in plants of the Anacardiaceae family, such as pistachio (Pistacia vera L.). Some AnAs have been associated with health benefits, and the position of the CC double bonds is a crucial feature of these metabolites. Herein, we propose a new strategy based on RPLC separation and detection by ESI-MS/MS, preceded by an epoxidation reaction. The procedure was applied to the green extracts of lignified pistachio shells, and a mixture of AnAs bearing alkyl chains 13:0, 15:0, and 17:1 emerged as prevailing. As positional isomers of AnA 15:1 (Δ8 and Δ6) and AnAs 17:1 (Δ10 and Δ8) were identified for the first time, their discovery paves the way to the systematic study of their potential health-beneficial effects. The developed method was validated and applied to quantify AnAs in pistachio ethanolic extract, showing contents higher than 10 mg/ 100 g of biomass.

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RATIONALE: Lyso derivatives of N-acyl-1,2-diacylglycero-3-phosphoethanolamines (L-NAPEs) are a lipid class mostly expressed in vegetables during stress and tissue damage that is involved in the synthesis of the lipid mediator N-acylethanolamines. L-NAPEs can be challenging to distinguish from isomeric phosphatidylethanolamines (PEs), especially in extracted complex samples where they could be confused with abundant PEs. METHODS: In this study, hydrophilic interaction liquid chromatography with electrospray ionization hyphenated with (tandem) mass spectrometry (MS) was proposed to distinguish L-NAPEs and PEs as deprotonated molecules, [M - H]─ , using both high-resolution/accuracy Fourier transform MS and low-resolution linear ion trap (LIT) mass analyzers. MS3 experiments of [M - H - KE]─ as precursor ions (KE, ketene loss) using the LIT instrument allowed us to distinguish between isomeric L-NAPE and PE species. RESULTS: Regiochemical rules were proposed working on enzymatically synthesized L-NAPEs. A few key differences in MS/MS spectra, including abnormal intensity of acyl chain losses as fatty acids, the presence of N-acylphosphoethanolamine ions, and diagnostic ions of the polar head, were disclosed. Additionally, MS3 spectra of [M - H - KE]─ as precursor ions allowed us to confirm the identification of L-NAPE species. The proposed rules were applied to samples extracted from tomato by-products including stems and leaves. CONCLUSIONS: Overall, our methodology is demonstrated as a robust approach to recognizing L-NAPEs in complex samples. L-NAPEs 18:2-N-18:2, 18:2-N-18:3, 18:3-N-18:2, and 18:2-N-18:1 were the prevailing compounds in the analyzed tomato samples, accounting for more than 90%. In summary, a reliable method for identifying L-NAPEs in complex samples is described. The proposed method could prevent overlooking L-NAPEs and overestimating isomeric PE species in future lipid analyses.

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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.

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Spirulina (Arthrospira platensis) proteins were extracted, digested, and analyzed by LC-ESI-FTMS/MS to find highly conserved peptides as markers of the microalga occurrence in foodstuffs. Putative markers were firstly chosen after in silico digestion of allergenic proteins, according to the FAO and WHO criteria, after assuring their presence in food supplements and in (un)processed foodsuffs. Parameters such as sensitivity, sequence size, and uniqueness for spirulina proteins were also evaluated. Three peptides belonging to C-phycocyanin beta subunit (P72508) were designated as qualifiers (ETYLALGTPGSSVAVGVGK and YVTYAVFAGDASVLEDR) and quantifier (ITSNASTIVSNAAR) marker peptides and used to validate the method for linearity, recovery, reproducibility, matrix effects, processing effects, LOD, and LOQ. The main aim was to determine spirulina in commercial foodstuffs like pasta, crackers, and homemade bread incurred with the microalga. The possible inclusion of the designated peptides in a standardized method, based on multiple reaction monitoring using a linear ion trap MS, was also demonstrated.

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A significant area of study and upgrading for increasing sensitivity and general performances of matrix-assisted laser-desorption ionization (MALDI) mass spectrometry (MS) is related to matrix design. Several efforts have been made to address the challenge of low-mass-region interference-free for metabolomics analysis and specifically for lipidomics. To this aim, rationally designed matrices as 4-chloro-α-cyanocinnamic acid (ClCCA) were introduced and reported to provide enhanced analytical performances. We have taken this rational design one step further by developing and optimizing new MALDI matrices with a range of modifications on the CHCA core, involving different functionalities and substituents. Of particular interest was the understanding of the electron-withdrawing (e.g., nitro-) or donating (e.g., methoxy-) effects along with the extent of conjugation on the ionization efficiency. In the present work, ten matrices were designed on a reasonable basis, synthesized, and characterized by NMR and UV spectroscopies and laser desorption ionization. With the assistance of these putative MALDI matrices, samples containing phospholipids (PL), and neutral di-/tri-acylglycerols (DAG, TAG) were investigated using milk, fish, blood, and human plasma extracts. In comparison with CHCA and ClCCA, four of them, viz. [(2E,4E)-2-cyano-5-(4-methoxyphenyl)penta-2,4-dienoic acid] (1), [(2E,4E)-2-cyano-5-(4-nitrophenyl)penta-2,4-dienoic acid] (2), [(E)-2-cyano-3-(6-methoxynaphthalen-2-yl)acrylic acid] (6) and [(E)-2-cyano-3-(naphthalen-2-yl)acrylic acid] (7) displayed good to even excellent performances as MALDI matrices in terms of ionization capability, interference-free spectra, S/N ratio, and reproducibility. Especially compound 7 (cyano naphthyl acrylic acid, CNAA) was the election matrix for PL analysis and matrix 2 (cyano nitrophenyl dienoic acid, CNDA) for neutral lipids such as DAG and TAG in positive ion mode.

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Water-soluble diacyl arsenosugar phospholipids (As-PL) are natural products widespread in marine animals and algae, including the brown alga Undaria pinnatifida, also known as wakame. The systematic recognition of As-PL has been hampered by the lack of standard and of qualitative methods to establish the carbon-carbon double bond positions of unsaturated fatty acyl chains. Here, the epoxidation reaction of fatty acyl substituents of As-PL was carried out with high selectivity by meta-chloroperoxybenzoic acid and the C-C double bond localization was established by collision-induced dissociation of epoxidized species as deprotonated molecules, [epoM - H]-. Reversed-phase liquid chromatography (RPLC) separation and a sequential triple-stage MS (i.e., MS3) analysis of unsaturated and epoxidized As-PL were very helpful to characterize the carbon-carbon double bond locations of both sn-1 and sn-2 fatty acyl chains, starting from a diagnostic product ion pair with 16.0 Da mass difference. These results indicate that intact As-PL can be annotated in terms of fatty acyl chain composition and in terms of their C-C double bond position(s). Interestingly, hexadecenoic (16:1 Δ9) and octadecenoic (18:1 Δ9) along with octadecadienoic (18:2 Δ9,12) and octadecatrienoic (18:3 Δ9,12,15) were found to be the most abundant unsaturated fatty acyl chains of As-PL in the brown alga wakame, thus confirming it as a good source of essential fatty acids with a balanced ω6/ω3 ratio. Although the toxicity of As-including metabolites of algal As-PL is still a matter of debate and needs to be studied in more detail, the described approach can be exploited to assess if As-PL could contribute to the supply of essential fatty acids related to the use of algae as nutritious food.

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Since novel nutrient sources with high protein content, such as yeast, fungi, bacteria, algae, and insects, are increasingly introduced in the consumer market, safety evaluation studies on their potentially allergenic proteins are required. A pipeline for in silico establishing the sequence-based homology between proteins of spirulina (Arthrospira platensis) and chlorella (Chlorella vulgaris) micro-algae and those included in the AllergenOnline (AO) database (AllergenOnline.org) is described. The extracted proteins were first identified through tryptic peptides analysis by reversed-phase liquid chromatography and high resolution/accuracy Fourier-transform tandem mass spectrometry (RPLC-ESI-FTMS/MS), followed by a quest on the UniProt database. The AO database was subsequently interrogated to assess sequence similarity between identified microalgal proteins and known allergens, based on criteria established by the World Health Organization (WHO) and Food and Agriculture Organization (FAO). A direct search for microalgal proteins already included in allergen databases was also performed using the Allergome database. Six proteins exhibiting a significant homology with food allergens were identified in spirulina extracts. Five of them, i.e., two thioredoxins (D4ZSU6, K1VP15), a superoxide dismutase (C3V3P3), a glyceraldehyde-3-phosphate dehydrogenase (K1W168), and a triosephosphate isomerase (D5A635), resulted from the search on AO. The sixth protein, C-phycocyanin beta subunit (P72508), was directly obtained after examining the Allergome database. Two proteins exhibiting significant sequence homology with food allergens were retrieved in chlorella extracts, viz. calmodulin (A0A2P6TFR8), which is related to troponin c (D7F1Q2), and fructose-bisphosphate aldolase (A0A2P6TDD0). Specific serum screenings based on immunochemical tests should be undertaken to confirm or rule out the allergenicity of the identified proteins.

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The lipidome of a brown seaweed commonly known as wakame (Undaria pinnatifida), which is grown and consumed around the world, including Western countries, as a healthy nutraceutical food or supplement, was here extensively examined. The study was focused on the characterization of phospholipids (PL) and glycolipids (GL) by liquid chromatography (LC), either hydrophilic interaction LC (HILIC) or reversed-phase LC (RPLC), coupled to electrospray ionization (ESI) and mass spectrometry (MS), operated both in high and in low-resolution mode. Through the acquisition of single (MS) and tandem (MS/MS) mass spectra more than 200 PL and GL of U. pinnatifida extracts were characterized in terms of lipid class, fatty acyl (FA) chain composition (length and number of unsaturations), and regiochemistry, namely 16 SQDG, 6 SQMG, 12 DGDG, 5 DGMG, 29 PG, 8 LPG, 19 PI, 14 PA, 19 PE, 8 PE, 38 PC, and 27 LPC. The FA (C16:0) was the most abundant saturated acyl chain, whereas the monounsaturated C18:1 and the polyunsaturated C18:2 and C20:4 chains were the prevailing ones. Odd-numbered acyl chains, iJ., C15:0, C17:0, C19:0, and C19:1, were also recognized. While SQDG exhibited the longest and most unsaturated acyl chains, C18:1, C18:2, and C18:3, in the sn-1 position of glycerol, they were preferentially located in the sn-2 position in the case of PL. The developed analytical approach might pave the way to extend lipidomic investigations also for other edible marine algae, thus emphasizing their potential role as a source of bioactive lipids.

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Glucuronic acid containing diacylglycerols (3-(O-α-d-glucuronopyranosyl)-1,2-diacyl-sn-glycerols, GlcA-DAG) are glycolipids of plant membranes especially formed under phosphate-depletion conditions. An analytical approach for the structural characterization of GlcA-DAG in red ripe tomato (Solanum lycopersicum L.) extracts, based on reversed-phase liquid chromatography (RPLC) coupled with electrospray ionization (ESI) and tandem mass spectrometry (MS/MS) using a linear ion trap, is described in this paper. At least 14 GlcA-DAG (R1/R2) species, including four regioisomers, containing three predominant fatty acyl chains C16:0, C18:2, and C18:3, were identified for the first time. Moreover, 29 GlcA-DAG acylated on the glucuronosyl ring (acyl-R3 GlcA-DAG) were discovered, alongside 15 acylated lyso-forms, i.e., acylated 3-(O-α-d-glucuronosyl)monoacylglycerols, abbreviated as acyl-R3 GlcA-MAG (R1/0) or (0/R2). Although many of these acylated lyso-forms were isomeric with GlcA-DAG (i.e., acyl chains with equivalent sum composition), they were successfully separated by reversed-phase liquid chromatography (RPLC) using a solid-core C18 column packed with 2.6 µm particle size. Tandem MS (and eventually MS3) data obtained from sodium adducts ([M + Na]+) and deprotonated molecules ([M - H]-) were fundamental to detect diagnostic product ions related to the glucuronosyl ring and then determine the identity of all investigated glycolipids, especially to recognize the acyl chain linked to the ring. A classification of GlcA-MAG, GlcA-DAG, and acylated GlcA-DAG and GlcA-MAG was generated by an in house-built database. The discovery of acylated derivatives emphasized the already surprising heterogeneity of glucuronic acid-containing mono- and diacylglycerols in tomato plants, stimulating interesting questions on the role played by these glycolipids.

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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.

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RATIONALE: Cisplatin (CP) is a widely used anticancer drug characterized by toxic side effects that could be alleviated using novel delivery systems including CP prodrugs. The in vitro incubation of a putative prodrug, obtained from cyanocobalamin (CNCbl) and cis-diamminemonochloroplatinum(II) (mCP), with nucleoside monophosphates (NMPs) was investigated. METHODS: The in vitro reactions between the putative prodrug CNCbl-mCP and the NMPs of adenosine (AMP), guanosine (GMP), cytidine (CMP) and uridine (UMP) were carried out in slightly acidic water-methanol solutions at 37°C for 24 h. Each sample was examined using reversed-phase liquid chromatography coupled with electrospray ionization in positive ion mode and tandem mass spectrometry (RPLC/ESI-MS/MS) by collision-induced dissociation in a linear ion-trap mass spectrometer. RESULTS: Seven adducts were recognized as formed by substitution reactions of the chloride ligand in planar CP. Comparison between observed and theoretical isotopic patterns together with MS/MS fragmentation pathways revealed the presence of single or multiple binding sites depending on the NMP involved. The CNCbl-mCP conjugate was found to interact with N7 or O4 atoms of GMP and UMP, respectively, generating single adducts, while two isomeric adducts were observed for CMP. Finally, AMP gave rise to three isomeric adducts. CONCLUSIONS: In agreement with literature data relevant to the interaction between CP and NMPs, the most reactive nucleotides were AMP and GMP. The present RPLC/ESI-MS/MS approach is very promising for investigation of the reactions of CP conjugates with ribonucleotides not only in vitro but also in vivo.

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Autism spectrum disorder (ASD) is a broad and heterogeneous group of neurological developmental disorders characterized by impaired social interaction and communication, restricted and repetitive behavioural patterns, and altered sensory processing. Currently, no reliable ASD molecular biomarkers are available. Since immune dysregulation has been supposed to be related with ASD onset and dyslipidaemia has been recognized as an early symptom of biological perturbation, lipid extracts from peripheral blood mononuclear cells (PBMCs), consisting primarily of lymphocytes (T cells, B cells, and NK cells) and monocytes, of 38 children with ASD and their non-autistic siblings were investigated by hydrophilic interaction liquid chromatography (HILIC) coupled with electrospray ionization and Fourier-transform mass spectrometry (ESI-FTMS). Performances of two freeware software for data extraction and processing were compared with acquired reliable data regardless of the used informatics. A reduction of variables from 1460 by the untargeted XCMS to 324 by the semi-untargeted Alex123 software was attained. All-ion fragmentation (AIF) MS scans along with Alex123 software were successfully applied to obtain information related to fatty acyl chain composition of six glycerophospholipid classes occurring in PBMC. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were explored to verify the occurrence of significant differences in the lipid pool composition of ASD children compared with 36 healthy siblings. After rigorous statistical validation, we conclude that phospholipids extracted from PBMC of children affected by ASD do not exhibit diagnostic biomarkers. Yet interindividual variability comes forth from this study as the dominant effect in keeping with the existing phenotypic and etiological heterogeneity among ASD individuals. Graphical abstract.

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