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Among the organic contaminants that could pass from waste to polyhydroxyalkanoates (PHAs), there are the polycyclic aromatic hydrocarbons (PAHs). For this reason, we have developed a rapid analytical method for the determination of sixteen PAHs in PHAs. PAHs were extracted by n-hexane, after matrix dispersion and crumbling into sand; the extract was purified by solid phase extraction using florisil as adsorbent. Recoveries in the range of 89–101% were obtained for the deuterated analytes, except for the two with the lowest molecular weight. Trueness between 92% and 108% and within-laboratory precision (expressed as relative standard deviation) ≤18% were estimated for all the analytes. Gas chromatography/mass spectrometry was used for analyte determination. Method limits of quantification were suitable to assure that PAH presence in PHA biolpolymers is much below the limits set by European law for plastic materials. Indeed, analysis of two different PHA samples showed that contamination is limited to few compounds at non-concerning levels.

The paper describes the development of an enrichment method for the analysis of the endogenous phosphopeptides in saliva. The method was based on magnetic solid phase extraction by a magnetic graphitized carbon black-TiO2 composite material and was developed considering different saliva pre-treatments, namely C18 solid phase extraction for purification, direct dilution in loading buffer or acetonitrile precipitation. The method was based on a shotgun proteomics workflow and the enriched peptide mixture was analysed by nanoHPLC and high resolution tandem mass spectrometry. Acetonitrile precipitation provided the best results, with up to 165 endogenous phosphopeptides identified in saliva samples from healthy individuals. The physico-chemical features of the identified endogenous phosphopeptides indicated that such peptides were large, hydrophilic and basic.

The paper describes the development of a targeted quantitative method for the analysis of policosanols in hemp inflorescence. Policosanols are long chain aliphatic alcohols, with carbon chains typically in the range 20–36, with interesting biological activities. These compounds are typically separated by gas chromatography and only a few methods employ liquid chromatography for policosanols. In both cases, methods always include the derivatization of policosanols. In this study, policosanols were separated by ultra-high performance liquid chromatography without any derivatization and detected using high resolution mass spectrometry by formation of lithiated adducts. The procedure was optimized and a quantitative method was validated for the most abundant policosanols (with C24, C26, C27, C28, and C30 chain lengths) in industrial hemp inflorescence extracts. The method was used for the quantitative analysis of policosanols in five hemp types. Hemp wax was found rich in these compounds, especially C26 and C28 policosanols, which may prove useful for revalorization of wax by-products. Finally, the acquired data were also used to expand the search to the untargeted qualitative analysis of policosanols using Compound Discoverer. The untargeted method allowed the annotation of underivatized policosanols up to C33.

The work describes the preparation of a new magnetic phase for batch enrichment of phosphopeptides. The material exploits the advantages of magnetic solid phase extraction and couples them with the most employed approach for phosphopeptide enrichment, i.e. Ti4+-IMAC. In order to immobilize Ti4+ ions on the surface of the magnetite nanoparticles, they were first covered by a silica shell and then modified to expose at the surface bromine containing groups. Glycidyl methacrylate was subsequently polymerized from these groups using the “grafting from” approach by the activator regenerated by electron transfer–atom transfer radical polymerization (ARGET-ATRP) technique. Finally, the glycidyl groups were reacted with iminodiacetic acid to functionalize the material with moieties suitable for coordination. The prepared material was extensively characterized and subsequently tested for enrichment of a bovine serum albumin mixture with casein to ascertain its potential. With positive results, the new magnetic polymeric material was further employed to set up an enrichment method on yeast protein digest based on shotgun proteomics. The sample to phase ratio was optimized and the best condition compared to a commercial TiO2 spin column. At the end of the comparison, the new material proved better and could enrich a larger total number of phosphopeptides with increased selectivity. All these conclusions and the test performed on a real complex sample within the final shotgun application further support the applicability of the new material in phosphopeptide analysis of real matrices.

Short peptides are of extreme interest in clinical and food research fields, nevertheless they still represent a crucial analytical issue. The main aim of this paper was the development of an analytical platform for a considerable advancement in short peptides identification. For the first time, short sequences presenting both natural and post-translationally modified amino acids were comprehensively studied thanks to the generation of specific databases. Short peptide databases had a dual purpose. First, they were employed as inclusion lists for a suspect screening mass-spectrometric analysis, overcoming the limits of data dependent acquisition mode and allowing the fragmentation of such low-abundance substances. Moreover, the databases were implemented in Compound Discoverer 3.0, a software dedicated to the analysis of short molecules, for the creation of a data processing workflow specifically dedicated to short peptide tentative identification. For this purpose, a detailed study of short peptide fragmentation pathways was carried out for the first time. The proposed method was applied to the study of short peptide sequences in enriched urine samples and led to the tentative identification more than 200 short natural and modified short peptides, the highest number ever reported.

Polyphenols are a broad class of plant secondary metabolites which carry out several biological functions for plant growth and protection and are of great interest as nutraceuticals for their antioxidant properties. However, due to their structural variability and complexity, the mass-spectrometric analysis of polyphenol content in plant matrices is still an issue. In this work, a novel approach for the identification of several classes of polyphenol derivatives based on ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry was developed. First, mass-spectrometric parameters were optimized in order to obtain a large set of diagnostic product ions for their high-confidence identification. The software Compound Discoverer 3.0 was then implemented with a comprehensive database of 45,567 polyphenol derivatives and with mass-spectrometric data for their building blocks, resulting in a specific tool for the semi-automatic identification of flavonoids, anthocyanins, ellagitannins, proanthocyanidins and phenolic acids. The method was then applied to the identification of polyphenols in industrial hemp (Cannabis sativa), a matrix whose use is recently spreading for pharmaceutical and nutraceutical purposes, resulting in the identification of 147 compounds belonging to the classes of flavonoids, proanthocyanidins and phenolic acids. The proposed method is applicable to the polyphenol profiling of any plant matrix and it is not dependent on data in the literature for their identification, allowing the discovery of compounds which have been never identified before.

When drug nanocarriers enter a physiological environment, their surface gets coated by a dynamic biomolecular corona (BMC) mainly constituted by proteins. Although a deep investigation has been performed on the composition of BMC in terms of proteins, scarce attention has been posed to low molecular weight metabolites present in human plasma. In this work, for the first time, the investigation of the BMC of liposomal nanoparticles (NPs) constituted by 1,2-dioleoyl-3-trimethylammonium-propane polar lipid has been carried out by an ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry based untargeted metabolomics approach. Compounds were tentatively identified based on matches with online databases and comparison of MS/MS spectra with available spectral libraries. Moreover, a comparison of three metabolite extraction strategies, including an ultrafiltration membrane based protocol, a methanol extraction based protocol, and Wessel & Flügge protocol, was performed. Methanol extraction procedure resulted in the widest metabolic coverage of liposomal NP BMC. A total of 193 metabolites has been tentatively identified, 166 of which belonged to the class of lipids including phospholipids, steroids, carnitines, fatty alcohols, diglycerides and fatty acids. The high abundance of lipids in the BMC can be explained by the adsorption of plasma lipoproteins onto liposome surface, confirming previous works on other kinds of NPs. Lipids are important bioactive molecules, which could impact NP circulation and uptake by cells. Extending the investigation of BMC beyond the protein corona and towards the "lipid corona" may be the keystone of a better understanding and control of NP fate in human body.

The untargeted profiling is a promising approach for the characterization of secondary metabolites in biological matrices. Thanks to the recent rapid development of high-resolution mass spectrometry (HRMS) instrumentations, the number of applications by untargeted approaches for biological samples profiling has widely increased in the recent years. Despite the high potentialities of HRMS, however, a major issue in natural products analysis often arises in the upstream process of compounds separation. A separation technique is necessary to avoid phenomena such as signal suppression, and it is especially needed in the presence of isomeric metabolites, which are otherwise indistinguishable. Glucosinolates (GLSs), a group of secondary metabolites widely distributed among plants, resulted to be associated to the prevention of some serious diseases, such as cancer. This led to the development of several methods for the analysis of GLSs in vegetables tissues. The issue of GLSs chromatographic separation has been widely studied in the past because of the difficulty in the analysis of this highly polar and variable class of compounds. Several alternatives to reversed phase (RP) chromatography, sometimes not compatible with the coupling of liquid chromatography with mass spectrometry, have been tested for the analysis of intact GLSs. However, the availability of new stationary phases, in the last years, could allow the re-evaluation of RP chromatography for the analysis of intact GLSs. In this work, a thorough evaluation of four RP chromatographic columns for the analysis of GLSs in cauliflower (Brassica oleracea L. var. botrytis) extracts by an ultra-high performance liquid chromatographic system coupled via electrospray source to a hybrid quadrupole-Orbitrap mass spectrometer is presented. The columns tested were the following: one column Luna Omega polar C18, one column Kinetex Biphenyl, one column Kinetex core-shell XB-C18, two columns Kinetex core-shell XB-C18. After a previous optimization of the extraction method, cauliflower extracts were analyzed testing four different mobile phases onto the four columns for a total of sixteen different chromatographic conditions. The chromatographic systems were evaluated based on the number of detected and tentatively identified GLSs. Luna Polar stationary phase resulted to be the most suitable for the analysis of GLSs compared to Kinetex XB and Kinetex Biphenyl columns stationary phase. However, two in series Kinetex XB columns increased the number of tentatively identified GLSs compared to one Kinetex XB, showing the importance of column length in the analysis of complex mixtures. The data obtained with the best chromatographic system were deeply analyzed by MS/MS investigation for the final identification. Fiflty-one GLSs were tentatively identified, 24 of which have never been identified in cauliflower. Finally the linearity of the analytes response over the analyzed range of concentration was checked, suggesting that the developed method is suitable for both qualitative and quantitative analysis of GLSs in phytochemical mixtures.

The analysis of the ascorbic acid (vitamin C; A.A.) contained in some foodstuff and pharmaceutical samples was performed by a new microcalorimetric method. It uses the oxidation of the vitamin C catalysed by the enzyme ascorbate oxidase (A.O.), which gets the specificity of the reaction. The calibration curve was built under the following operative conditions: 25.00 degrees C, pH 5.6, [A.O.]=11 IU ml(-1), the linearity range is: 3/=A.A./=270 mg l(-1). The method has been applied to many samples to determine the vitamin C quantity. Each analysis takes about 20 min (the assay time is instead about 5 min, the slow step is due to the time necessary for the calorimeter to return to the operative temperature after washing). No pre-treatments of the samples are requested, unless their solubilisation in buffer. The variations of vitamin C concentration were studied in function of storage time and light exposition. Using a known HPLC assay to compare its reliability on a drug analysis has validated the proposed method.

Phytoestrogens are diphenolic compounds that are present in several edible plants and are particularly abundant in soybeans. Because of their estrogenical, antriestrogenical, anticarcinogenic and antioxidant activities in animal and humans, they became of great interest. Dietary factors are considered important in determination of risks, in fact, studies have revealed beneficial or protective effects of the consumption of vegetables, in particular soy and soybean products. So that in the present paper the simultaneous determination of eight isoflavones and coumestrol in vegetables is reported. The quantitative analysis has been made by means of LC separation combined with tandem mass spectrometry. In particular, a new simple and fast extraction methodology and a clean-up, based on cold aided de-fatting, is proposed. Method performance was evaluated by comparison with a reference procedure. The developed procedure was then used for a survey of phytoestrogens concentration in some selected vegetables.

Many enzymatic reactions yield volatile products either directly or by cascade sequences, so it seems possible that head-space chromatography might be used to determine enzymatic activity. The activity of urinary kallikrein, as arginine esterase, has been determined in this way by using the Nα-acetyl- l -phenylalanyl- l -arginine ethyl ester as substrate and measuring the ethanol yielded on incubation for 10 min at 30°, followed by quenching of the reaction. The method has been applied to aqueous solutions and urine.

Analysis for 13 common organophosphorus pesticides by thin-layer chromatography is described; 17 solvent systems were examined. With channel thin-layer chromatography, linear calibration graphs were obtained for the range 1–10 μg.

A number of resins containing ethylenediamine acetic acid groups have been prepared, and these intermediates (resin-EDTA) converted into the iron(III) form. The capacities of these exchangers in the formation of iron(III)-phenol complexes have been studied and compared with those of the Chelex-iron(III) resin. The character of the exchanger matrix is very important in connection with the retention of phenols and with the elution order. The modified Amberlite CG 4B in the iron(III) form can be used for the quantitative separation of phenolic compounds, a separation that is not possible with the Chelex-iron(III) resin.

The properties of a chelating ion-exchanger in the iron(III) form in contact with an aqueous solution containing phenolic compounds have been studied. The phenols retained are completely displaced by elution with an ethanolic sodium hydroxide solution.