First Proof of Concept of a Click Inverse Electron Demand Diels-Alder Reaction for Assigning the Regiochemistry of Carbon-Carbon Double Bonds in Untargeted Lipidomics.

Lipidomics by high-resolution mass spectrometry (HRMS) has become a prominent tool in clinical chemistry due to the proven connections between lipid dysregulation and the insurgence of pathologies. However, it is difficult to achieve structural characterization beyond the fatty acid level by HRMS, especially when it comes to the regiochemistry of carbon-carbon double bonds, which play a major role in determining the properties of cell membranes. Several approaches have been proposed for elucidating the regiochemistry of double bonds, such as derivatization before MS analysis by photochemical reactions, which have shown great potential for their versatility but have the unavoidable drawback of splitting the MS signal. Among other possible approaches for derivatizing electron-rich double bonds, the emerging inverse-electron-demand Diels-Alder (IEDDA) reaction with tetrazines stands out for its unmatchable kinetics and has found several applications in basic biology and protein imaging. In this study, a catalyst-free click IEDDA reaction was employed for the first time to pinpoint carbon-carbon double bonds in free and conjugated fatty acids. Fatty acid and glycerophospholipid regioisomers were analyzed alone and in combination, demonstrating that the IEDDA reaction had click character and allowed the obtention of diagnostic product ions following MS/MS fragmentation as well as the possibility of performing relative quantitation of lipid regioisomers. The IEDDA protocol was later employed in an untargeted lipidomics study on plasma samples of patients suffering from prostate cancer and benign prostatic conditions, confirming the applicability of the proposed reaction to complex matrices of clinical interest.

Preparation of Monolith for Online Extraction and LC-MS Analysis of ß-Estradiol in Serum Via a Simple Multicomponent Reaction.

Multicomponent reactions offer efficient and environmentally friendly strategies for preparing monoliths suitable for applications in analytical chemistry. In the described study, a multicomponent reaction was utilized for the one-pot miniaturized preparation of a poly(propargyl amine) polymer inside commercial silica-lined PEEK tubing. The reaction involved only small amounts of reagents and was characterized by atom economy. The resulting monolithic column was incorporated into an autosampler system for the online extraction and cleanup of ß-estradiol from human serum. Sample pretreatment was simplified to a simple dilution with methanol and centrifugation to remove proteins. The resulting platform included LC-MS analysis in multiple reaction monitoring for quantitative analysis of ß-estradiol. The method was validated in serum, demonstrating practical applicability for the monitoring of fertile women. Recoveries were above 94%, and LOD and LOQ values at 0.008 and 0.18 ng mL-1, respectively. The developed platform proved to be competitive with previous methods for solid-phase microextraction of ß-estradiol in serum, with comparable recovery and sensitivity but with the advantage of nearly complete automation. The environmental impact of the process was evaluated as acceptable due to the miniaturization of the monolith synthesis and the automation of extraction. The drawback associated with the LC-MS technique can be reduced by the inclusion of additional analytes in a single investigation. The work demonstrates that multicomponent reactions are versatile, economical, and possibly a green methodology for producing reversed-phase and mixed-mode sorbents, enabling miniaturization of the entire analytical procedure from the preparation of extraction sorbents to analysis.

One-phase extraction coupled with photochemical reaction allows the in-depth lipid characterization of hempseeds by untargeted lipidomics.

Due to their valuable nutritional content, several hemp-derived products from hempseeds have recently been placed in the market as food and food ingredients. In particular, the lipid composition of hempseeds has raised interest for their rich content in biologically active polyunsaturated fatty acids with an optimum ratio of omega-3 and omega-6 compounds. At present, however, the overall polar lipidome composition of hempseeds remains largely unknown. In the present work, an analytical platform was developed for the extraction, untargeted HRMS-based analysis, and detailed annotation of the lipid species. First, five one- and two-phase solid-liquid extraction protocols were tested and compared on a hempseed pool sample to select the method that allowed the overall highest efficiency as well as easy coupling with lipid derivatization by photochemical [2 + 2] cycloaddition with 6-azauracil. Underivatized lipids were annotated employing a data processing workflow on Compound Discoverer software that was specifically designed for polar lipidomics, whereas inspection of the MS/MS spectra of the derivatized lipids following the aza-Paternò-Büchi reaction allowed pinpointing the regiochemistry of carbon-carbon double bonds. A total of 184 lipids were annotated, i.e., 26 fatty acids and 158 phospholipids, including minor subclasses such as N-acylphosphatidylethanolamines. Once the platform was set up, the lipid extracts from nine hempseed samples from different hemp strains were characterized, with information on the regiochemistry of free and conjugated fatty acids. The overall analytical approach helped to fill a gap in the knowledge of the nutritional composition of hempseeds.

Dispersive solid phase extraction using a hydrophilic molecularly imprinted polymer for the selective extraction of patulin in apple juice samples.

A molecularly imprinted polymer with a specific selectivity for patulin was successfully synthesized. The molecularly imprinted material was prepared using the two functional monomers dopamine and melamine and formaldehyde as the cross-linker. The resulting material possessed a large number of hydrophilic groups, such as hydroxyls, imino groups, and ether linkages. For the first time, uric acid was used as a dummy template for its structural similarity to patulin. Comprehensive characterization and detailed studies of the adsorption process were carried out via adsorption isotherms, while the rate-limiting steps were investigated using adsorption kinetics. Separation, determination, and quantification of patulin were achieved by ultra-high performance liquid chromatography coupled with both photodiode array detection and tandem mass spectrometry. The latter was applied to patulin confirmation in the analysis of real samples. The methodology was validated in 20 apple juice samples. The results showed that the developed hydrophilic molecularly imprinted polymer had high selectivity and specific adsorption towards patulin, with mean recoveries ranging between 85 and 90% and a relative standard deviation lower than 15%. The developed molecularly imprinted polymer exhibited good linearity in the range 1-100 ng mL-1 with coefficient of determination (R2) > 0.99. The limit of detection was 0.5 ng mL-1, and the limit of quantification was 1 ng g-1. The developed method showed a good purification capacity for apple juices due to its hydrophilic nature and the polar interactions established with the target analyte.

Untargeted cannabinomics reveals the chemical differentiation of industrial hemp based on the cultivar and the geographical field location.

Cannabis sativa has long been harvested for industrial applications related to its fibers. Industrial hemp cultivars, a botanical class of Cannabis sativa with a low expression of intoxicating Δ9-tetrahydrocannabinol (Δ9-THC) have been selected for these purposes and scarcely investigated in terms of their content in bioactive compounds. Following the global relaxation in the market of industrial hemp-derived products, research in industrial hemp for pharmaceutical and nutraceutical purposes has surged. In this context, metabolomics-based approaches have proven to fulfill the aim of obtaining comprehensive information on the phytocompound profile of cannabis samples, going beyond the targeted evaluation of the major phytocannabinoids. In the present paper, an HRMS-based metabolomics study was addressed to seven distinct industrial hemp cultivars grown in four experimental fields in Northern, Southern, and Insular Italy. Since the role of minor phytocannabinoids as well as other phytocompounds was found to be critical in discriminating cannabis chemovars and in determining its biological activities, a comprehensive characterization of phytocannabinoids, flavonoids, and phenolic acids was carried out by LC-HRMS and a dedicated data processing workflow following the guidelines of the metabolomics Quality Assurance and Quality Control Consortium. A total of 54 phytocannabinoids, 134 flavonoids, and 77 phenolic acids were annotated, and their role in distinguishing hemp samples based on the geographical field location and cultivar was evaluated by ANOVA-simultaneous component analysis. Finally, a low-level fused model demonstrated the key role of untargeted cannabinomics extended to lesser-studied phytocompound classes for the discrimination of hemp samples.

Occurrence of per- and polyfluorinated alkyl substances in wastewater treatment plants in Northern Italy.

Wastewater treatment plants are known to be relevant input sources of per- and polyfluoroalkyl substances (PFAS) in the aquatic environment. This study aimed to investigate the occurrence, fate, and seasonal variability of twenty-five PFAS in four municipal wastewater treatment plants (WWTP A, B, C, and D) surrounding the city of Milan (Northern, Italy). Composite 24-h wastewater samples were collected in July and October 2021 and May and February 2022 from influents and effluents of the four WWTPs. PFAS were detected at concentrations ranging between 24.1 and 66.9 µg L-1 for influent and 13.4 and 107 µg L-1 for effluent wastewater samples. Perfluoropentanoic acid was the most abundant (1.91-30.0 µg L-1) in influent samples, whereas perfluorobutane sulfonic acid predominated (0.80-66.1 µg L-1) in effluent samples. In sludge, PFOA was detected in plant A at concentrations in the range of 96.6-165 ng kg-1 dw in primary sludge samples and 98.6-440 ng kg-1 dw in secondary treatment sludge samples. The removal efficiency of total PFAS varied between 6 % and 96 %. However, an increase of PFAS concentrations was observed from influents to effluents for plant D (during July and October), plant A (during October and May), and plant C (during May) indicating that biotransformation of PFAS precursors can occur during biological treatments. This was supported by the observed increase in concentrations of PFOA from primary to secondary treatment sludge samples in plant A. Moreover, the plant operating at shorter hydraulic retention times (plant D) showed lower removal efficiency (<45 %). Seasonal variation of PFAS in influent and effluent appears rather low and more likely due to pulse release instead of seasonal factors.

Recent trends in glycoproteomics by characterization of intact glycopeptides.

This trends article provides an overview of the state of the art in the analysis of intact glycopeptides by proteomics technologies based on LC-MS analysis. A brief description of the main techniques used at the different steps of the analytical workflow is provided, giving special attention to the most recent developments. The topics discussed include the need for dedicated sample preparation for intact glycopeptide purification from complex biological matrices. This section covers the common approaches with a special description of new materials and innovative reversible chemical derivatization strategies, specifically devised for intact glycopeptide analysis or dual enrichment of glycosylation and other post-translational modifications. The approaches are described for the characterization of intact glycopeptide structures by LC-MS and data analysis by bioinformatics for spectra annotation. The last section covers the open challenges in the field of intact glycopeptide analysis. These challenges include the need of a detailed description of the glycopeptide isomerism, the issues with quantitative analysis, and the lack of analytical methods for the large-scale characterization of glycosylation types that remain poorly characterized, such as C-mannosylation and tyrosine O-glycosylation. This bird's-eye view article provides both a state of the art in the field of intact glycopeptide analysis and open challenges to prompt future research on the topic.

Biotic transformation products of sulfonamides in environmental water samples: High-resolution mass spectrometry-based tentative identification by a suspect screening approach.

The presence of pharmaceuticals in the aquatic environment is mainly due to their release from the effluents of the wastewater treatment plants (WWTPs), which are unable to completely remove them and their transformation products (TPs). Sulfonamides (SAs) are a synthetic antibacterial class used for the treatment of both human and animal infections; they have often been reported in surface water, thus contributing to the antibiotic resistance emergency. Monitoring SA TPs should be important as well because they could still exert some pharmaceutical activity; however, many TPs are still unknown since several transformation processes are possible (e. g. human and animal metabolism, WWTP activities, environmental factors etc.). In this work, three of the most used SAs, i.e., sulfamethoxazole (SMX), sulfapyridine (SPY), and sulfadiazine (SDZ), were incubated for 20 days in a batch reactor with activated sludge under controlled conditions. Then, the water sample was extracted and analyzed by ultra-high performance liquid chromatography-high resolution mass spectrometry in the data dependent acquisition (DDA) mode. Starting from the literature data, the possible transformation pathways were studied, and for each SA, a list of TPs was hypothesized and used for the identification. The raw data files were processed with Compound Discoverer, and 44 TPs (18, 13, and 13 TPs for SMX, SPY, and SDZ, respectively), including multiple TPs, were manually validated. To overcome the limitation of the DDA, the identified TPs were used in an inclusion list to analyze WWTP samples by a suspect screening approach. In this way, 4 SMX TPs and 5 SPY TPs were tentatively identified together with their parent compounds. Among these TPs, 5 of 9 were acetylated forms, in agreement with previous literature reporting that acetylation is the predominant SA transformation.

New hydrophilic material based on hydrogel polymer for the selective enrichment of intact glycopeptides from serum protein digests.

The paper describes the preparation and characterization of a new HILIC material for the enrichment of N-linked glycopeptides. The material was prepared using 2-acrylamido-2-methyl-1-propanesulfonic acid as the monomer and ethylene glycol dimethacrylate as the cross-linker. The material was developed by a Box-Behnken experimental design, taking into consideration the amount of monomer-to-crosslinker ratio, the composition, and the amount of porogen mixture. By this approach, the property of the resulting polymer could be fine-tuned to modulate the hydrophilicity and porosity. As HILIC enrichment is mostly dependent on hydrophilic interactions, including H-bonding, the amount of swelling was expected to have an important function, therefore the optimization considered a monomer percent in the range of 20-80%, which implied very different water swelling capacities. After assessing the potential of this new polymer family on fetuin digests, the 17 materials resulting from the Box-Behnken experimental design were used for the enrichment of glycopeptides from serum protein digests. The materials displayed a superior performance over cotton HILIC enrichment, both in terms of the number of enriched N-linked glycopeptides and selectivity, providing up to 762 N-linked glycopeptides with 77% selectivity. The optimization indicated that a high amount of monomer significantly affected the number of enriched glycopeptides, which is also closely connected with the hydrogel nature of the resulting polymers. The results not only provide one additional HILIC material for the enrichment of glycopeptides but also pave the way for the use and development of hydrogel materials for the enrichment of N-linked glycopeptides.

Evaluation of the carotenoid and fat-soluble vitamin profile of industrial hemp inflorescence by liquid chromatography coupled to mass spectrometry and photodiode-array detection.

Industrial hemp (Cannabis sativa L.) is a plant matrix whose use is recently spreading for pharmaceutical and nutraceutical purposes. Detailed characterization of hemp composition is needed for future research that further exploits the beneficial effects of hemp compounds on human health. Among minor constituents, carotenoids and fat-soluble vitamins have largely been neglected to date despite carrying out several biological activities and regulatory functions. In the present paper, 22 target carotenoids and fat-soluble vitamins were analyzed in the inflorescences of seven Italian industrial hemp varieties cultivated outdoor. The analytes were extracted by cold saponification to avoid artifacts and analyzed by high-performance liquid chromatography coupled with Selected reaction monitoring mass spectrometry. Phytoene, phytofluene, and all-trans-ß-carotene were the most abundant in all analyzed samples (31-55 µg g-1, 11.6-29 µg g-1, and 7.3-53 µg g-1, respectively). Besides the target analytes, liquid chromatography coupled with photodiode-array detection allowed us to tentatively identify several other carotenoids based on their retention behavior and UV-vis spectra with the support of theoretical rules and data in the literature. To the best of our knowledge, this is the first comprehensive characterization of carotenoids and fat-soluble vitamins in industrial hemp inflorescence.

Isolation and functional characterization of hemp seed protein-derived short- and medium-chain peptide mixtures with multifunctional properties for metabolic syndrome prevention.

This study aims to obtain a valuable mixture of short-chain peptides from hempseed as a new ingredient for developing nutraceutical and functional foods useful for preventing metabolic syndrome that represents the major cause of death globally. A dedicated analytical platform based on a purification step by size exclusion chromatography or ultrafiltration membrane and high-resolution mass spectrometry was developed to isolate and comprehensively characterize short-chain peptides leading to the identification of more than 500 short-chain peptides. Our results indicated that the short-chain peptide mixture was about three times more active than the medium-chain peptide mixture and total hydrolysate with respect to measured inhibition of the angiotensin-converting enzyme. The short-chain peptide mixture was also two times more active as a dipeptidyl peptidase IV, and twofold more active on the cholesterol metabolism pathway through the modulation of low-density lipoprotein receptor.

Untargeted analysis of environmental contaminants in surface snow samples of Svalbard Islands by liquid chromatography-high resolution mass spectrometry.

In recent years, there is increasing attention on the contaminants of emerging concern (CECs), which include plasticizers, flame retardants, industrial chemicals, pharmaceuticals, and personal care products, since they have been detected even far away from pollution sources. The polar regions are not exempt from the presence of anthropogenic contaminants, and they are employed as a model for understanding the pollutant fate and impact. During the 2021 spring campaign, sixteen surface snow samples were collected close to the research station of Ny-Ålesund located on the Spitsbergen Island of the Norwegian Svalbard Archipelago. The samples were extracted by solid-phase extraction and analyzed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS) following an untargeted approach. Compound tentative identification was obtained with the aid of the software Compound Discoverer, using both mass spectral database search and manual validation. Among the 114 compounds identified with a high confidence level in the snow samples, >80 have some commercial or industrial use (drugs, plasticizers, fragrances, etc.), therefore they could be of anthropogenic origin. Nonetheless, a clear contamination trend did not appear in the snow samples collected on eight different days during one month. The comparison with aerosol samples collected in the same area did not help identifying the source, either, since only a few compounds were in common, and they were mainly of natural origin. As such, the analysis of aerosol sample did not support possible long-range transport, also considering that compounds were detected mostly in the coarse fraction.

Novel Aza-Paternò-Büchi Reaction Allows Pinpointing Carbon-Carbon Double Bonds in Unsaturated Lipids by Higher Collisional Dissociation.

The evaluation of double bond positions in fatty acyl chains has always been of great concern given their significance in the chemical and biochemical role of lipids. Despite being the foremost technique for lipidomics, it is difficult in practice to obtain identification beyond the fatty acyl level by the sole high-resolution mass spectrometry. Paternò-Büchi reactions of fatty acids (FAs) with ketones have been successfully proposed for pinpointing double bonds in FAs in combination with the collision-induced fragmentation technique. In the present paper, an aza-Paternò-Büchi (aPB) reaction of lipids with 6-azauracil (6-AU) was proposed for the first time for the determination of carbon-carbon double bonds in fatty acyl chains using higher collisional dissociation in the negative ion mode. The method was optimized using free FA and phospholipid analytical standards and compared to the standard Paternò-Büchi reaction with acetone. The introduction of the 6-AU moiety allowed enhancing the ionization efficiency of the FA precursor and diagnostic product ions, thanks to the presence of ionizable sites on the derivatizing agent. Moreover, the aPB derivatization allowed the obtention of deprotonated ions of phosphatidylcholines, thanks to an intramolecular methyl transfer from the phosphocholine polar heads during ionization. The workflow was finally applied for pinpointing carbon-carbon double bonds in 77 polar lipids from an yeast (Saccharomyces cerevisiae) extract.

Insights into the syntrophic microbial electrochemical oxidation of toluene: a combined chemical, electrochemical, taxonomical, functional gene-based, and metaproteomic approach.

Biodegradation of aromatic hydrocarbons in anoxic contaminated environments is typically limited by the lack of bioavailable electron acceptors. Microbial electrochemical technologies (METs) are able to provide a virtually inexhaustible electron acceptor in the form of a solid electrode. Recently, we provided first experimental evidence for the syntrophic degradation of toluene in a continuous-flow bioelectrochemical reactor known as the "bioelectric well". Herein, we further analyzed the structure and function of the electroactive toluene-degrading microbiome using a suite of chemical, electrochemical, phylogenetic, proteomic, and functional gene-based analyses. The bioelectric well removed 83 ± 7 % of the toluene from the influent with a coulombic efficiency of 84 %. Cyclic voltammetry allowed to identify the formal potentials of four putative electron transfer sites, which ranged from -0.2 V to +0.1 V vs. SHE, consistent with outer membrane c-type cytochromes and pili of electroactive Geobacter species. The biofilm colonizing the surface of the anode was indeed highly enriched in Geobacter species. On the other hand, the planktonic communities thriving in the bulk of the reactor harbored aromatic hydrocarbons degraders and fermentative propionate-producing microorganisms, as revealed by phylogenetic and proteomic analyses. Most likely, propionate, acetate or other VFAs produced in the bulk liquid from the degradation of toluene were utilized as substrates by the electroactive biofilm. Interestingly, key-functional genes related to the degradation of toluene were found both in the biofilm and in the planktonic communities. Taken as a whole, the herein reported results highlight the importance of applying a comprehensive suite of techniques to unravel the complex cooperative metabolisms occurring in METs.

Stereoselective Synthesis of Spiro-Decalin Oxindole Derivatives via Sequential Organocatalytic Michael-Domino Michael/Aldol Reaction.

A highly stereoselective procedure for the synthesis of spiro-polycyclic oxindoles bearing five contiguous stereogenic centers including two tetrasubstituted carbons has been developed. Under sequential organocatalysis performed by a pyrrolidine-based organocatalyst and DBU, a highly atom-economical Michael-domino Michael/aldol reaction sequence was optimized, yielding variously functionalized spiro-decalin oxindoles with excellent stereoselectivity (>99:1 dr, up to 92% ee).

The Key Role of Metal Adducts in the Differentiation of Phosphopeptide from Sulfopeptide Sequences by High-Resolution Mass Spectrometry.

Site localization of protein sulfation by high-throughput proteomics remains challenging despite the technological improvements. In this study, sequence analysis and site localization of sulfation in tryptic peptides were determined under a conventional nano-liquid chromatography-mass spectrometry configuration. Tryptic sulfopeptide standards were used to study different fragmentation strategies, including collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), electron-transfer dissociation (ETD), electron-transfer/higher-energy collision dissociation (EThcD), and electron-transfer/collision-induced dissociation (ETciD), in the positive ionization mode. Sulfopeptides displayed only neutral loss of SO3 under CID, while the sequence could be determined for all other tested fragmentation techniques. Results were compared to the same sequences with phosphotyrosine, indicating important differences, as the sequence and modification localization could be studied by all fragmentation strategies. However, the use of metal adducts, especially potassium, provided valuable information for sulfopeptide localization in ETD and ETD-hybrid strategies by stabilizing the modification and increasing the charge state of sulfopeptides. In these conditions, both the sequence and localization could be obtained. In-source neutral loss of SO3 under EThcD provided diagnostic peaks suitable to distinguish the sulfopeptides from the nearly isobaric phosphopeptides. Further confirmation on the modification type was found in the negative ionization mode, where phosphopeptides always had the typical phosphate product ion corresponding to PO3-.

Investigating the Short Peptidome Profile of Italian Dry-Cured Ham at Different Processing Times by High-Resolution Mass Spectrometry and Chemometrics.

Short peptides have been spiking interest owing to their significant contribution to the taste and functional properties of dry-cured ham. In this study, a suspect screening approach based on high-resolution mass spectrometry was employed for the comprehensive characterization of the short endogenous peptidome in dry-cured ham samples at different processing stages (14, 22, and 34 months). After careful manual spectra interpretation, a chemometric approach based on principal component analysis was employed for highlighting the differences between the three sets of samples. A total of 236 short peptide sequences was tentatively identified, including 173 natural short peptides and 63 sequences containing non-proteinogenic amino acids, the highest number ever reported for endogenous sequences in dry-cured ham. Samples in the latest processing stages presented a generally higher abundance of dipeptides, indicating residual proteolytic activity. Moreover, the several annotated modified short peptides, mainly pyroglutamination and lactoyl conjugation, allowed hypothesizing several reactions occurring over time. For the first time, several lactoyl-dipeptides were tentatively identified in dry-cured ham samples with maximum concentration in the late processing stage samples. The presented results significantly contribute to the understanding of the reaction involving short peptides that affect the sensory and functional properties of dry-cured ham.

Comprehensive biomarker profiles and chemometric filtering of urinary metabolomics for effective discrimination of prostate carcinoma from benign hyperplasia.

Prostate cancer (PCa) is the most commonly diagnosed cancer in male individuals, principally affecting men over 50 years old, and is the leading cause of cancer-related deaths. Actually, the measurement of prostate-specific antigen level in blood is affected by limited sensitivity and specificity and cannot discriminate PCa from benign prostatic hyperplasia patients (BPH). In the present paper, 20 urine samples from BPH patients and 20 from PCa patients were investigated to develop a metabolomics strategy useful to distinguish malignancy from benign hyperplasia. A UHPLC-HRMS untargeted approach was carried out to generate two large sets of candidate biomarkers. After mass spectrometric analysis, an innovative chemometric data treatment was employed involving PLS-DA classification with repeated double cross-validation and permutation test to provide a rigorously validated PLS-DA model. Simultaneously, this chemometric approach filtered out the most effective biomarkers and optimized their relative weights to yield the highest classification efficiency. An unprecedented portfolio of prostate carcinoma biomarkers was tentatively identified including 22 and 47 alleged candidates from positive and negative ion electrospray (ESI+ and ESI-) datasets. The PLS-DA model based on the 22 ESI+ biomarkers provided a sensitivity of 95 ± 1% and a specificity of 83 ± 3%, while that from the 47 ESI- biomarkers yielded an 88 ± 3% sensitivity and a 91 ± 2% specificity. Many alleged biomarkers were annotated, belonging to the classes of carnitine and glutamine metabolites, C21 steroids, amino acids, acetylcholine, carboxyethyl-hydroxychroman, and dihydro(iso)ferulic acid.

Detailed investigation of the composition and transformations of phenolic compounds in fresh and fermented Vaccinium floribundum berry extracts by high-resolution mass spectrometry and bioinformatics.

INTRODUCTION: Blueberries are known for their very high content of biologically active phenolic compounds; nonetheless, differently from the North American and European species of blueberries, Neotropical blueberries have not been extensively studied yet. OBJECTIVES: In the present paper, the phenolic composition of Vaccinium floribundum Kunth, which is endemic to the Andean regions and grows 1,600 to 4,500 meters above sea level, was investigated by ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). Native and fermented berries were compared in terms of phenolic composition as well as antioxidant activity, total phenolic content, and total anthocyanin content. MATERIALS AND METHODS: V. floribundum native and fermented berries were extracted and analyzed by UHPLC-HRMS. The acquired datasets were processed by Compound Discoverer 3.1 using a dedicated data analysis workflow that was specifically set up for phenolic compound identification. RESULTS: In total, 309 compounds were tentatively identified, including anthocyanins, flavonoids, phenolic acids, and proanthocyanidins. The molecular transformations of phenolic compounds during fermentation were comprehensively investigated for the first time, and by a customized data processing workflow, 13 quinones and quinone methides were tentatively identified in the fermented samples. Compared to other species of the genus Vaccinium, a peculiar phenolic profile is observed, with low abundance of highly methylated compounds. CONCLUSION: Andean berries are a rich source of a wide variety of phenolic compounds. Untargeted MS analyses coupled to a dedicated data processing workflow allowed expanding the current knowledge on these berries, improving our understanding of the fate of phenolic compounds after fermentation.

Fully Automatized Detection of Phosphocholine-Containing Lipids through an Isotopically Labeled Buffer Modification Workflow.

High-resolution mass spectrometry is the foremost technique for qualitative and quantitative lipidomics analyses. Glycerophospholipids and sphingolipids, collectively termed polar lipids, are commonly investigated by hyphenated liquid chromatography-mass spectrometry (LC-MS) techniques that reduce aggregation effects and provide a greater dynamic range of detection sensitivity compared to shotgun lipidomics. However, automatic polar lipid identification is hindered by several isobaric and isomer mass overlaps, which cause software programs to often fail to correctly annotate the lipid species. In the present paper, a buffer modification workflow based on the use of labeled and unlabeled acetate ions in the chromatographic buffers was optimized by Box-Behnken design of the experiments and applied to the characterization of phosphocholine-containing lipids in human plasma samples. The contemporary generation of [M + CH3COO]-, [M + CD3COO]-, and [M - CH3]- coupled with a dedicated data processing workflow, which was specifically set up on Compound Discoverer software, allowed us to correctly determine adduct composition, molecular formulas, and grouping, as well as granting a lower false-positive rate and streamlining the manual validation step compared to commonly employed lipidomics platforms. The proposed workflow represents a robust yet easier alternative to the existing approaches for improving lipid annotation, as it does not require extensive sample pretreatment or prior isotopic enrichment or derivatization.