Lipidomics reveals the reshaping of the mitochondrial phospholipid profile in cells lacking OPA1 and mitofusins.

Depletion or mutations of key proteins for mitochondrial fusion, like optic atrophy 1 (OPA1) and mitofusins 1 and 2 (Mfn 1 and 2), are known to significantly impact the mitochondrial ultrastructure, suggesting alterations of their membranes' lipid profiles. In order to make an insight into this issue, we used hydrophilic interaction liquid chromatography coupled with electrospray ionization-high resolution MS to investigate the mitochondrial phospholipid (PL) profile of mouse embryonic fibroblasts knocked out for OPA1 and Mfn1/2 genes. One hundred sixty-seven different sum compositions were recognized for the four major PL classes of mitochondria, namely phosphatidylcholines (PCs, 63), phosphatidylethanolamines (55), phosphatidylinositols (21), and cardiolipins (28). A slight decrease in the cardiolipin/PC ratio was found for Mfn1/2-knockout mitochondria. Principal component analysis and hierarchical cluster analysis were subsequently used to further process hydrophilic interaction liquid chromatography-ESI-MS data. A progressive decrease in the incidence of alk(en)yl/acyl species in PC and phosphatidylethanolamine classes and a general increase in the incidence of unsaturated acyl chains across all the investigated PL classes was inferred in OPA1 and Mfn1/2 knockouts compared to WT mouse embryonic fibroblasts. These findings suggest a reshaping of the PL profile consistent with the changes observed in the mitochondrial ultrastructure when fusion proteins are absent. Based on the existing knowledge on the metabolism of mitochondrial phospholipids, we propose that fusion proteins, especially Mfns, might influence the PL transfer between the mitochondria and the endoplasmic reticulum, likely in the context of mitochondria-associated membranes.

Analysis of surfactants by mass spectrometry: Coming to grips with their diversity.

Surfactants are surface-active agents widely used in numerous applications in our daily lives as personal care products, domestic, and industrial detergents. To determine complex mixtures of surfactants and their degradation products, unselective and rather insensitive methods, based on colorimetric and complexometric analyses are no longer employable. Analytical methodologies able to determine low concentration levels of surfactants and closely related compounds in complex matrices are required. The recent introduction of robust, sensitive, and selective mass spectrometry (MS) techniques has led to the rapid expansion of the surfactant research field including complex mixtures of isomers, oligomers, and homologues of surfactants as well as their chemically and biodegradation products at trace levels. In this review, emphasis is given to the state-of-the-art MS-based analysis of surfactants and their degradation products with an overview of the current research landscape from traditional methods involving hyphenate techniques (gas chromatography-MS and liquid chromatography-MS) to the most innovative approaches, based on high-resolution MS. Finally, we outline a detailed explanation on the utilization of MS for mechanistic purposes, such as the study of micelle formation in different solvents.

Mass spectrometric characterization of aminophospholipids containing N-(2-hydroxyethyl)glycine in kombu algae extracts.

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.

Development of a New Binary Matrix for the Comprehensive Analysis of Lipids and Pigments in Micro- and Macroalgae Using MALDI-ToF/ToF Mass Spectrometry.

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.

Matrix Selection Strategies for MALDI-TOF MS/MS Characterization of Cyclic Tetrapyrroles in Blood and Food Samples.

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.

Food allergen detection by mass spectrometry: From common to novel protein ingredients.

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.

Oxidative NHC Catalysis: An Unconventional Tool for Macrocyclic Oligoesters Synthesis.

The application of N-heterocyclic carbene (NHC) catalysis under highly diluted oxidative condition to the polycondensation of dialdehydes and diols is herein presented as an alternative, atom-economical synthetic route to macrocyclic oligoesters (MCOs). The disclosed protocol paves the way to the straightforward access to MCOs, starting from commercial dialdehydes, avoiding the use of toxic diacyl chlorides, commonly employed in traditional MCOs synthetic processes. The method is totally metal-free, takes place in the green Me-THF solvent and requires the use of a fully recyclable quinone oxidant. The protocol versatility is confirmed by the employment of fossil-based and bio-based monomers such as 2,5-diformylfuran (DFF), 2,5-bis(hydroxymethyl)furan (BHMF), and isomannide, synthesizing a series of novel and known synthetically relevant macrocyclic oligoesters, fully characterized by NMR and MALDI-TOF MS analysis, with product yields (51-86 %) comparable to those obtained by traditional synthetic routes. Finally, to emphasize the synthetic relevance of the target macrocycles, an entropically-driven ring opening polymerization (ED-ROP) key study has been performed, optimizing the organocatalyzed synthesis of poly(2,5-furan-dimethylene 2,5 furandicarboxylate) (PBHMF) with number-average molecular weight up to 8200 g mol-1 and 66 % isolated yield.

PE, or not PE, that is the question: The case of overlooked lyso-N-acylphosphatidylethanolamines.

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.

The Effect of Milling on the Ethanolic Extract Composition of Dried Walnut (Juglans regia L.) Shells.

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.

The Mycotoxin Patulin Inhibits the Mitochondrial Carnitine/Acylcarnitine Carrier (SLC25A20) by Interaction with Cys136 Implications for Human Health.

The effect of mycotoxin patulin (4-hydroxy-4H-furo [3,2c] pyran-2 [6H] -one) on the mitochondrial carnitine/acylcarnitine carrier (CAC, SLC25A20) was investigated. Transport function was measured as [3H]-carnitineex/carnitinein antiport in proteoliposomes reconstituted with the native protein extracted from rat liver mitochondria or with the recombinant CAC over-expressed in E. coli. Patulin (PAT) inhibited both the mitochondrial native and recombinant transporters. The inhibition was not reversed by physiological and sulfhydryl-reducing reagents, such as glutathione (GSH) or dithioerythritol (DTE). The IC50 derived from the dose-response analysis indicated that PAT inhibition was in the range of 50 µM both on the native and on rat and human recombinant protein. The kinetics process revealed a competitive type of inhibition. A substrate protection experiment confirmed that the interaction of PAT with the protein occurred within a protein region, including the substrate-binding area. The mechanism of inhibition was identified using the site-directed mutagenesis of CAC. No inhibition was observed on Cys mutants in which only the C136 residue was mutated. Mass spectrometry studies and in silico molecular modeling analysis corroborated the outcomes derived from the biochemical assays.

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.

Synthesis and Investigation of Novel CHCA-Derived Matrices for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometric Analysis of Lipids.

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.

Editorial to the Special Issue "Lipidomics and Neurodegenerative Diseases".

The contribution of dysregulation of lipid signaling and metabolism to neurodegenerative diseases including Alzheimer's and Parkinson's is the focus of this special issue. Here, the matter of three reviews and one research article is summarized.

Lipidomics of the Edible Brown Alga Wakame (Undaria pinnatifida) by Liquid Chromatography Coupled to Electrospray Ionization and Tandem Mass Spectrometry.

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.

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.

In Situ Hydrogel Extraction with Dual-Enzyme Digestion of Proteinaceous Binders: the Key for Reliable Mass Spectrometry Investigations of Artworks.

A novel strategy based on in situ dual-enzyme digestion of paint layer proteinaceous binders is introduced for faster and more confident identification, resulting in a bottom-up proteomics approach by MALDI-TOF mass spectrometry (MS). In situ sampling/extraction of proteinaceous binders using small pieces of a hydrophilic gel, previously loaded with trypsin and chymotrypsin proteolytic enzymes, was successfully exploited. Along with minimal invasiveness, the synergy of both enzymes was very useful to increase the number of annotated peptide peaks with their corresponding amino acid sequence by database search and subsequent MALDI-TOF/TOF analysis. The protocol was initially aimed at enhancing the identification of egg-based binders and then validated on fresh and aged model pictorial layers; an increased protein coverage was significantly attained regardless of the used painting binders. Optical microscope images and spectrophotocolorimetry analysis evidenced that the painting layers were not damaged or altered because of contact/sampling without leaving hydrogel residues. The proposed protocol was successfully applied on a painted altarpiece "Assumption of the Virgin" dated to the XVI century and on an angel statue of the Nativity crib dated to the XII century, both from Altamura's Cathedral (Apulia, Italy). The occurrence of various protein binders of animal origin was easily and reliably ascertained.

Directional Immobilization of Proteins on Gold Nanoparticles Is Essential for Their Biological Activity: Leptin as a Case Study.

Gold nanomaterials hold great potential for biomedical applications. While this field is evolving rapidly, little attention has been paid to precise nanoparticle design and functionalization. Here, we show that when using proteins as targeting moieties, it is fundamental to immobilize them directionally to preserve their biological activity. Using full-length leptin as a case study, we have developed two alternative conjugation strategies for protein immobilization based on either a site-selective or a nonselective derivatization approach. We show that only nanoparticles with leptin immobilized site-selectively fully retain the ability to interact with the cognate leptin receptor. These results demonstrate the importance of a specified molecular design when preparing nanoparticles labeled with proteins.

In vitro reactions of a cyanocobalamin-cisplatin conjugate with nucleoside monophosphates.

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.

Coceth sulfate characterization by electrospray ionization tandem mass spectrometry.

RATIONALE: The anionic surfactants, among which are alkyl ether sulfates (AESs), are the most used class of surfactants in cleansing applications. The negatively charged head group of AESs is a sulfate moiety linked with a variable number of ethylene oxide units, i.e. a polyethylene glycol chain. The hydrophobic part of an AES is constituted by a linear alkyl chain of carbon atoms, generally obtained from natural fatty acids. Coconut oil fatty acids, including the sodium salts of coceth sulfate (CES) with chemical formula Cx Hy (OCH2 CH2 )n OSO3 Na, are widely used as feedstock for AESs synthesis. CES is added to many cleaning products and detergents defined as non-aggressive. Currently, no detailed structural information concerning the alkyl chain length x and, more importantly, the degree of ethoxylation n has been reported. METHODS: A commercial standard solution of CES was characterized by tandem mass spectrometry, employing direct injection into the electrospray ionization (ESI) source of a a linear quadrupole ion trap mass spectrometer. RESULTS: Two series of oligomeric species, characterized by a C12 and C14 alkyl chains, i.e. [C12 H25 (OCH2 CH2 )nOSO3 ]- and [C14 H29 (OCH2 CH2 )n OSO3 ]- with n ranging from 0 to 7, were successfully identified. The interpretation of these data was very useful for CES identification in three commercial dishwasher cleaning products. CONCLUSIONS: Direct injection MS/MS analysis of CES revealed a well-defined molecular weight distribution and allowed the alkyl chain composition and the number of ethylene oxide units to be to identified.

Profiling of quercetin glycosides and acyl glycosides in sun-dried peperoni di Senise peppers (Capsicum annuum L.) by a combination of LC-ESI(-)-MS/MS and polarity prediction in reversed-phase separations.

Interest in targeted profiling of quercetin glycoconjugates occurring in edible foodstuffs continues to expand because of their recognized beneficial health effects. Quercetin derivatives encompass several thousands of chemically distinguishable compounds, among which there are several compounds with different glycosylations and acylations. Since reference standards and dedicated databases are not available, the mass spectrometric identification of quercetin glycoconjugates is challenging. A targeted liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS) was applied for screening quercetin glycoconjugates in edible peperoni di Senise peppers (Capsicum annuum L.), protected by the European Union with the mark PGI (i.e., Protected Geographical Indication), and cultivated in Basilicata (Southern Italy). Chromatographic separation was accomplished by reversed-phase liquid chromatography (RPLC) using water/acetonitrile as the mobile phase and detection was performed on a linear ion trap mass spectrometer fitted with an electrospray ionization (ESI) source operating in negative ion mode. A correlation between experimental RP chromatographic retention time and those predicted by partition coefficients (log P) along with MS/MS data and an in-house developed database (named QUEdb) provided deep coverage for sixteen quercetin glycoconjugates. Among them, eleven quercetin glycoconjugates were already described in the literature and five were reported for the first time. These last acyl glycosidic quercetin derivatives were tentatively identified as quercetin-(galloyl-rhamnoside)-hexoside, [C34H33O20]- at m/z 761.1; quercetin-(sinapoyl-hexoside)-rhamnoside, [C38H39O20]- at m/z 815.4; quercetin-(galloyl-caffeoyl-hexoside)-rhamnoside, [C43H39O23]- at m/z 923.0; quercetin-(feruloyl-hexoside)-rhamnoside, [C37H37O19]- at m/z 785.1; and quercetin-(succinyl-rhamnoside)-rhamnoside, [C31H33O18]- at m/z 693.1. Graphical abstract.