Quantification of glucuronidated and sulfated steroids in human urine by ultra-high pressure liquid chromatography quadrupole time-of-flight mass spectrometry

The urinary steroid profile is constituted by anabolic androgenic steroids, including testosterone and its relatives, that are extensively metabolized into phase II sulfated or glucuronidated steroids. The use of liquid chromatography coupled to mass spectrometry (LC-MS) is an issue for the direct analysis of conjugated steroids, which can be used as urinary markers of exogenous steroid administration in doping analysis, without hydrolysis of the conjugated moiety. In this study, a sensitive and selective ultra high-pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometer (UHPLC-QTOF-MS) method was developed to quantify major urinary metabolites simultaneously after testosterone intake. The sample preparation of the urine (1 mL) was performed by solid-phase extraction on Oasis HLB sorbent using a 96-well plate format. The conjugated steroids were analyzed by UHPLC-QTOF-MS^E with a single-gradient elution of 36 min (including re-equilibration time) in the negative electrospray ionization mode. MS^E analysis involved parallel alternating acquisitions of both low- and high-collision energy functions. The method was validated and applied to samples collected from a clinical study performed with a group of healthy human volunteers who had taken testosterone, which were compared with samples from a placebo group. Quantitative results were also compared to GC-MS and LC-MS/MS measurements, and the correlations between data were found appropriate. The acquisition of full mass spectra over the entire mass range with QTOF mass analyzers gives promise of the opportunity to extend the steroid profile to a higher number of conjugated steroids.     

Essential Oil of Cannabis sativa L: Comparison of Yield and Chemical Composition of 11 Hemp Genotypes

Cannabis sativa L. is an annual species cultivated since antiquity for different purposes. While, in the past, hemp inflorescences were considered crop residues, at present, they are regarded as valuable raw materials with different applications, among which extraction of the essential oil (EO) has gained increasing interest in many fields. The aim of the present study is the evaluation of the yield and the chemical composition of the EO obtained by hydrodistillation from eleven hemp genotypes, cultivated in the same location for two consecutive growing seasons. The composition of the EOs was analyzed by GC–MS, and then subjected to multivariate statistical analysis. Sesquiterpenes represented the main class of compounds in all the EOs, both in their hydrocarbon and oxygenated forms, with relative abundances ranging from 47.1 to 78.5%; the only exception was the Felina 32 sample collected in 2019, in which cannabinoids predominated. Cannabinoids were the second most abundant class of compounds, of which cannabidiol was the main one, with relative abundances between 11.8 and 51.5%. The statistical distribution of the samples, performed on the complete chemical composition of the EOs, evidenced a partition based on the year of cultivation, rather than on the genotype, with the exception of Uso-31. Regarding the extraction yield, a significant variation was evidenced among both the genotypes and the years of cultivation.     

De Novo Design,Synthesis and Characterisation of MP3, A New Catalytic Four‐Helix Bundle Hemeprotein

A new artificial metalloenzyme, MP3 (MiniPeroxidase 3), designed by combining the excellent structural properties of four‐helix bundle protein scaffolds with the activity of natural peroxidases, was synthesised and characterised. This new hemeprotein model was developed by covalently linking the deuteroporphyrin to two peptide chains of different compositions to obtain an asymmetric helix–loop–helix/heme/helix–loop–helix sandwich arrangement, characterised by 1) a His residue on one chain that acts as an axial ligand to the iron ion; 2) a vacant distal site that is able to accommodate exogenous ligands or substrates; and 3) an Arg residue in the distal site that should assist in hydrogen peroxide activation to give an HRP‐like catalytic process. MP3 was synthesised and characterised as its iron complex. CD measurements revealed the high helix‐forming propensity of the peptide, confirming the appropriateness of the model procedure; UV/Vis, MCD and EPR experiments gave insights into the coordination geometry and the spin state of the metal. Kinetic experiments showed that Fe^III–MP3 possesses peroxidase‐like activity comparable to R38A–hHRP, highlighting the possibility of mimicking the functional features of natural enzymes. The synergistic application of de novo design methods, synthetic procedures, and spectroscopic characterisation, described herein, demonstrates a method by which to implement and optimise catalytic activity for an enzyme mimetic.     

Charge-sensitive vibrational modes in the (EDT-TTF-OX)2AsF6 chiral molecular conductors

Infrared and Raman spectra of three chiral molecular conductors (EDT-TTF-OX)₂AsF₆, comprising of two salts based on enantiopure EDT-TTF-OX donor molecules and one based on their racemic mixture, have been measured as a function of temperature. In the frequency range of the C=C stretching vibrations of EDT-TTF-OX, charge-sensitive modes are identified based on theoretical calculations for neutral and oxidized EDT-TTF-OX using density functional theory (DFT) methods. The positions of C=C stretching modes in both Raman and infrared spectra of the (EDT-TTF-OX)₂AsF₆ materials are analyzed assuming a linear relationship between the frequency and charge of the molecule. The charge density on the EDTTTF-OX donor molecule is estimated to be +0.5 in all investigated materials and does not change with temperature. Therefore we suggest, that M-I transition observed in (EDT-TTF-OX)₂AsF₆ chiral molecular conductors at low temperature is not related to the charge ordering mechanism.     

Twists and turns in the hierarchical self-assembly pathways of a non-amphiphilic chiral supramolecular material

The formation of helical self-assembled fibres by a C(3) symmetric molecule incorporating three tetrathiafulvalene units is shown to be influenced dramatically by the processing conditions, leading to a variety of different chiral forms, including unprecedented croissants.     

Antimicrobial Activity of Silver and Gold Nanoparticles Prepared by Photoreduction Process with Leaves and Fruit Extracts of Plinia cauliflora and Punica granatum

The increased number of resistant microbes generates a search for new antibiotic methods. Metallic nanoparticles have emerged as a new platform against several microorganisms. The nanoparticles can damage the bacteria membrane and DNA by oxidative stress. The photoreduction process is a clean and low-cost method for obtaining silver and gold nanoparticles. This work describes two original insights: (1) the use of extracts of leaves and fruits from a Brazilian plant Plinia cauliflora, compared with a well know plant Punica granatum, and (2) the use of phytochemicals as stabilizing agents in the photoreduction process. The prepared nanoparticles were characterized by UV-vis, FTIR, transmission electron microscopy, and Zeta potential. The antimicrobial activity of nanoparticles was obtained with Gram-negative and Gram-positive bacteria, particularly the pathogens Staphylococcus aureus ATCC 25923; Bacillus subtilis ATCC 6633; clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecalis; Escherichia coli ATCC 25922; Escherichia coli O44:H18 EAEC042 (clinical isolate); Klebsiella pneumoniae ATCC 700603, Salmonella Thiphymurium ATCC 10231; Pseudomonas aeruginosa ATCC 27853; and Candida albicans ATCC 10231. Excellent synthesis results were obtained. The AgNPs exhibited antimicrobial activities against Gram-negative and Gram-positive bacteria and yeast (80–100%), better than AuNPs (0–87.92%), and may have the potential to be used as antimicrobial agents.     

Biodeterioration of Paper: A SEM Study of Fungal Spoilage Reproduced Under Controlled Conditions

Biodeterioration phenomena represent a complex of physical and chemical alteration processes in various materials, such as those constituting the objects that represent our cultural heritage. The biodegradation of paper is conditioned by several variables such as the materials from which cellulose is obtained, the manufacturing processes employed, the occurrence of other affecting substances such as lignin or metallic compounds, and by the environmental conditions in which papers are conserved. In this study, biodeterioration of paper was artificially induced in order to evaluate the role of a range of chemical and physical variables on damage caused by cellulolytic fungi. A variable pressure SEM instrument was used to characterise paper samples with different fibre origins, and alterations obtained in vitro. Two fungal strains, Aspergillus terreus Thom and Chaetomium globosum Kunze, which are cellulolytic species frequently associated with paper spoilage, were used to produce stains with characteristics close to those observable on art objects made from paper. The stains obtained on the different samples of paper were compared at both low and high magnification, in order to visualize the macro- and microscopic characteristics of paper fibres, inorganic constituents, impurities, and the deteriorating agents related to the spoiled areas. During this survey it was observed that single paper characteristics can strongly influence the intensity and the results of the fungal action. For example, the activity of a fungal strain on paper grades containing fibres of the same origin, but with different sizing, led to the formation of profoundly different stains and alterations. Moreover fungal structures, analysed by low vacuum SEM, in areas on paper corresponding to the stains appeared in different physiological states suggesting an important effect of paper constituents on fungal growth and their sporulating ability.     

Adulteration of diesel/biodiesel blends by vegetable oil as determined by Fourier transform (FT) near infrared spectrometry and FT-Raman spectroscopy

In this work it has been shown that the routine ASTM methods (ASTM 4052, ASTM D 445, ASTM D 4737, ASTM D 93, and ASTM D 86) recommended by the ANP (the Brazilian National Agency for Petroleum, Natural Gas and Biofuels) to determine the quality of diesel/biodiesel blends are not suitable to prevent the adulteration of B2 or B5 blends with vegetable oils. Considering the previous and actual problems with fuel adulterations in Brazil, we have investigated the application of vibrational spectroscopy (Fourier transform (FT) near infrared spectrometry and FT-Raman) to identify adulterations of B2 and B5 blends with vegetable oils. Partial least square regression (PLS), principal component regression (PCR), and artificial neural network (ANN) calibration models were designed and their relative performances were evaluated by external validation using the F-test. The PCR, PLS, and ANN calibration models based on the Fourier transform (FT) near infrared spectrometry and FT-Raman spectroscopy were designed using 120 samples. Other 62 samples were used in the validation and external validation, for a total of 182 samples. The results have shown that among the designed calibration models, the ANN/FT-Raman presented the best accuracy (0.028%, w/w) for samples used in the external validation.     

Determination of Bitterness of Extra Virgin Olive Oils by Amperometric Detection

A flow injection system with amperometric detection at potentials poised at +0.4 and +0.9 V was used to evaluate intensity of the bitter taste in monovarietal Extra Virgin Olive Oils (EVOO). Results from the proposed method were based on the extraction of the bitter constituents of the virgin olive oil samples in methanol‐water, followed by the direct amperometric measurement. These potentials were selected according to the hydrodynamic voltammogram of oleuropein, one of the most prominent and bitter phenolic compound found in EVOO. The amperometric detection was applied on 32 monovariatal EVOO samples. Results were correlated with the phenolic profile measured by high performance liquid chromatography (HPLC). The amperometric signal at +0.9 V mainly correlated with the total phenols of the samples (R²=0.81), whereas the signal at +0.4 V mainly correlated with oleuropein aglycone (3,4 DHPEA‐EDA, R²=0.79). Bitterness intensity of the samples was evaluated by a trained sensory panel of experts and the results compared to those obtained by the amperometric flow system. The best correlation with the bitter taste was achieved by the sensor at +0.4 V (R²=0.72). A calibration model based on partial least squares was built with three variables, namely the sensors set at +0.4 and +0.9 V and the total phenol content of the EVOO extracts. The model showed a moderate capacity to predict the bitterness of the EVOO samples using leave one out method, (R²=0.75) and in prediction of a test set of samples (R²=0.7). Such approach is very promising for future studies.     

Cafiero and Brooks–Jewett theorems for Vitali spaces

We introduce a common generalization of Boolean rings and lattice ordered groups called Vitali spaces and we give a version of Cafiero and Brooks–Jewett convergence Theorems for additive functions defined in a Vitali space with values in a Hausdorff commutative topological group. This article was supported by MURST, project “Analisi Reale” and by GNAMPA of Istituto Nazionale di Alta Matematica.