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.

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.

Characterization of the Trans-Epithelial Transport of Green Tea (C. sinensis) Catechin Extracts with In Vitro Inhibitory Effect against the SARS-CoV-2 Papain-like Protease Activity.

This work describes an untargeted analytical approach for the screening, identification, and characterization of the trans-epithelial transport of green tea (Camellia sinensis) catechin extracts with in vitro inhibitory effect against the SARS-CoV-2 papain-like protease (PLpro) activity. After specific catechin extraction, a chromatographic separation obtained six fractions were carried out. The fractions were assessed in vitro against the PLpro target. Fraction 5 showed the highest inhibitory activity against the SARS-CoV-2 PLpro (IC50 of 0.125 µg mL-1). The untargeted characterization revealed that (-)-epicatechin-3-gallate (ECG) was the most abundant compound in the fraction and the primary molecule absorbed by differentiated Caco-2 cells. Results indicated that fraction 5 was approximately 10 times more active than ECG (IC50 value equal to 11.62 ± 0.47 µg mL-1) to inhibit the PLpro target. Overall, our findings highlight the synergistic effects of the various components of the crude extract compared to isolated ECG.

Untargeted Characterization of Chestnut (Castanea sativa Mill.) Shell Polyphenol Extract: A Valued Bioresource for Prostate Cancer Cell Growth Inhibition.

Chestnut seeds are used for fresh consumption and for the industrial preparation of derivatives, such as chestnut flour. During industrial processing, large amounts of by-products are generally produced, such as leaves, flowers, shells and burs. In the present study, chestnut shells were extracted by boiling water in order to obtain polyphenol-rich extracts. Moreover, for the removal or non-phenolic compounds, a separation by preparative reverse phase chromatography in ten fractions was carried out. The richest fractions in terms of phenolic content were characterized by means of untargeted high-resolution mass spectrometric analysis together with a dedicated and customized data processing workflow. A total of 243 flavonoids, phenolic acids, proanthocyanidins and ellagitannins were tentatively identified in the five richest fractions. Due its high phenolic content (450.03 µg GAE per mg of fraction), one tumor cell line (DU 145) and one normal prostate epithelial cell line (PNT2) were exposed to increasing concentration of fraction 3 dry extract for 24, 48 and 72 h. Moreover, for DU 145 cell lines, increase of apoptotic cells and perturbation of cell cycle was demonstrated for the same extract. Those outcomes suggest that chestnut industrial by-products could be potentially employed as a source of bioresources.

Peptidomic Approach for the Identification of Peptides with Potential Antioxidant and Anti-Hyperthensive Effects Derived From Asparagus By-Products.

Asparagus waste represents products of great interest since many compounds with high biological value are located in the lower portion of the spears. The extraction of bioactive compounds from asparagus by-products is therefore crucial for the purpose of adding value to these by-products. In this paper, bioactive peptides from asparagus waste were extracted, digested, purified and identified. In particular, Alcalase® was chosen as the enzyme to use to obtain protein hydrolysate due to its low cost and, consequently, the possibility of implementing the method on a large scale. In order to simplify the peptide extract to reach better identification, the hydrolysate was fractionated by reversed-phase chromatography in 10 fractions. Two tests were carried out for antioxidant activity (ABTS-DPPH) and one for antihypertensive activity (ACE). Fractions with a higher bioactivity score were identified by peptidomics technologies and screened for bioactivity with the use of bioinformatics. For ACE-inhibitor activity, two peptides were synthetized, PDWFLLL and ASQSIWLPGWL, which provided an EC50 value of 1.76 µmol L-1 and 4.02 µmol L-1, respectively. For the antioxidant activity, by DPPH assay, MLLFPM exhibited the lowest EC50 value at 4.14 µmol L-1, followed by FIARNFLLGW and FAPVPFDF with EC50 values of 6.76 µmol L-1 and 10.01 µmol L-1, respectively. A validation of the five identified peptides was also carried out. The obtained results showed that peptides obtained from asparagus by-products are of interest for their biological activity and are suitable for being used as functional ingredients.

Investigation of free and conjugated seleno-amino acids in wheat bran by hydrophilic interaction liquid chromatography with tandem mass spectrometry.

An analytical method for determining seleno-methionine, methyl-seleno-cysteine, and seleno-cystine in wheat bran was developed and validated. Four different extraction procedures were evaluated to simultaneously extract endogenous free and conjugated seleno-amino acids in wheat bran in order to select the best extraction protocol in terms of seleno amino acid quantitation. The extracted samples were subjected to a clean-up by a reversed phase/strong cation exchange solid-phase extraction and analyzed by chiral hydrophilic interaction liquid chromatography-tandem mass spectrometry. The optimized extraction protocol was employed to validate the methodology. Process efficiency ranged from 58 to 112% and trueness from 73 to 98%. Limit of detection and limit of quantification were lower than 1 ng/g. Four wheat bran samples were analyzed for both total Se and single seleno-amino acids determination. The results showed that Se- seleno-methyl-lselenocysteine was the major seleno-amino acid in wheat bran while seleno-methionine and seleno-cysteine were both minor species.

Delving into the Polar Lipidome by Optimized Chromatographic Separation, High-Resolution Mass Spectrometry, and Comprehensive Identification with Lipostar: Microalgae as Case Study.

The work describes the chromatographic separation optimization of polar lipids on Kinetex-EVO, particularly focusing on sulfolipids in spirulina microalgae ( Arthrospira platensis). Gradient shape and mobile-phase modifiers (pH and buffer) were tested on lipid standards. Different conditions were evaluated, and resolution, peak capacity, and peak shape were calculated both in negative mode, for sulfolipids and phospholipids, and in positive mode, for glycolipids. A high-confidence lipid identification strategy was also applied. In collaboration with software creators and developers, Lipostar was implemented to improve the identification of phosphoglycerolipids and to allow the identification of glycosylmonoradyl- and glycosyldiradyl-glycerols classes, the last being the main focus of this work. By this approach, an untargeted screening also for searching lipids not yet reported in the literature could be accomplished. The optimized chromatographic conditions and database search were tested for lipid identification first on the standard mixture, then on the polar lipid extract of spirulina microalgae, for which 205 lipids were identified.

Analytical strategies based on chromatography-mass spectrometry for the determination of estrogen-mimicking compounds in food.

Food safety can be compromised by the presence of a wide variety of substances, deriving from both natural and anthropogenic sources. Among these substances, compounds exhibiting various degrees of estrogenic activity have been widely studied in environmental samples, whereas less attention has been devoted to food matrices. The aim of the present review is to give a general overview on the recent analytical methods based on gas or liquid chromatography coupled to mass spectrometry for the determination of estrogen-like compounds in foods, including new developments, improvements and upcoming trends in the field. Attention will be focused on four representative groups of compounds, i.e. natural and synthetic estrogens, mycoestrogens, phytoestrogens, and alkylphenols.