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Chlorella vulgaris is a popular microalga used for biofuel production; nevertheless, it possesses a strong cell wall that hinders the extraction of molecules, especially lipids within the cell wall. For tackling this issue, we developed an efficient and cost‐effective method for optimal lipid extraction. Microlaga cell disruption by acid hydrolysis was investigated comparing different temperatures and reaction times; after hydrolysis, lipids were extracted with n‐hexane. The best recoveries were obtained at 140°C for 90 min. The microalgae were then analyzed by an untargeted approach based on liquid chromatography with high‐resolution mass spectrometry, providing the tentative identification of 28 fatty acids. First, a relative quantification on the untargeted data was performed using peak area as a surrogate of analyte abundance. Then, a targeted quantitative method was validated for the tentatively identified fatty acids, in terms of recovery (78‐100%), intra‐ and interday relative standard deviations ( 0.98). The most abundant fatty acids were palmitic, palmitoleic, oleic, linoleic, linolenic, and stearic acids.

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.

An analytical method for determining seleno-methionine (SeMet), methyl-seleno-cysteine and seleno-cystine in extra-virgin olive oil (EVOO) was developed and validated. EVOO sample (15 g) was diluted with hexane, extracted with methanol/water 80:20 (v/v), and cleaned up by a reversed phase/strong cation exchange solid phase extraction. Analysis was performed by chiral hydrophilic interaction liquid chromatography-tandem mass spectrometry. Process efficiency ranged between 49 and 97% and trueness between 87 and 126%, with intermediate precision, expressed as standard deviation, lower than 10%. Method detection limits (MDLs) and method quantification limits (MQLs) were lower than 1 μg kg−1. Thirty-two EVOO samples from different Italian regions were analyzed for both total Se and single seleno-amino acids determination. Only l -SeMet was found at level MQL (0.2 μg kg−1)-1.42 μg kg−1 in ten samples, while total Se was in the range of MDL-9.1 μg kg−1. Concentration of l -SeMet (5–6% of total Se) and total Se correlated very well to each other (R2 = 0.995).

This review is a collection of recent applications of mass spectrometry studies for the characterization of phytocannabinoids in cannabis and hemp plant material and related products. The focus is mostly on recent applications using mass spectrometry as detector, in hyphenation to typical separation techniques (i.e., liquid chromatography or gas chromatography), but also with less common couplings or by simple direct analysis. The papers are described starting from the most common approach for targeted quantitative analysis, with applications using low-resolution mass spectrometry equipment, but also with the introduction of high-resolution mass analyzers as the detectors. This reflects a common trend in this field, and introduces the most recent applications using high-resolution mass spectrometry for untargeted analysis. The different approaches used for untargeted analysis are then described, from simple retrospective analysis of compounds without pure standards, through untargeted metabolomics strategies, and suspect screening methods, which are the ones currently allowing to achieve the most detailed qualitative characterization of the entire phytocannabinoid composition, including minor compounds which are usually overlooked in targeted studies and in potency evaluation. These approaches also represent powerful strategies to answer questions on biological and pharmacological activity of cannabis, and provide a sound technology for improved classification of cannabis varieties. Finally, open challenges are discussed for future directions in the detailed study of complex phytocannabinoid mixtures.

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.

In this work, an untargeted approach based on ultra-high performance liquid chromatography coupled to high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) was employed for the analysis of contaminants in surface water samples, possibly deriving from an ineffective removal by wastewater treatment plants or illicit release. First, solid-phase extraction (SPE) conditions for 30 target compounds were optimized for two different sorbents, namely the commercial Oasis HLB and graphitized carbon black (GCB), by Box-Behnken designs of experiments. Upon identification of optimal conditions, the two SPE materials yielded comparable extraction recoveries, ranging between 52-110% and 55-100% for Oasis HLB and GCB, respectively. Thereafter, river water samples collected in five locations near Rome (Italy) were extracted using both sorbents and then analyzed by UHPLC-HRMS/MS in both positive and negative electrospray (ESI) ion polarities. The retrieved raw data files were submitted to Compound Discoverer software and compound identification was tentatively assigned by matching their tandem mass spectra with the mzCloud mass spectral library and manual validation, resulting in a total of 241 identified compounds (211 and 40 in positive and negative ESI mode, respectively), mainly belonging to pharmaceuticals.

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.

The work describes the development of an enrichment method for the analysis of endogenous phosphopeptides in serum. Endogenous peptides can play significant biological roles, and some of them could be exploited as future biomarkers. In this context, blood is one of the most useful biofluids for screening, but a systematic investigation of the endogenous peptides, especially phosphorylated ones, is still lacking, mainly due to the lack of suitable analytical methods. Thus, in this paper, different phosphopeptide enrichment strategies were pursued, based either on metal oxide affinity chromatography (MOAC, in the form of commercial TiO2 spin columns or magnetic graphitized carbon black-TiO2 composite), or on immobilized metal ion affinity chromatography (IMAC, in the form of Ti4+-IMAC magnetic material or commercial Fe3+-IMAC spin columns). While MOAC strategies proved completely unsuccessful, probably due to interfering phospholipids displacing phosphopeptides, the IMAC materials performed very well. Different sample preparation strategies were tested, comprising direct dilution with the loading buffer, organic solvent precipitation, and lipid removal from the matrix, as well as the addition of phosphatase inhibitors during sample handling for maximized endogenous phosphopeptide enrichment. All data were acquired by a shotgun peptidomics approach, in which peptide samples were separated by reversed-phase nanoHPLC hyphenated with high-resolution tandem mass spectrometry. The devised method allowed the identification of 176 endogenous phosphopeptides in fresh serum added with inhibitors by the direct dilution protocol and the Ti4+-IMAC magnetic material enrichment, but good results could also be obtained from the commercial Fe3+-IMAC spin column adapted to the batch enrichment protocol.

A sensitive and straightforward HPLC-UV method was developed for the simultaneous quantification of the two main impurities in “pure” commercial cannabigerol (CBG) samples. The identification of such impurities, namely cannabigerovarin (CBGV) and cannabigerobutol (CBGB), the propyl and butyl homologs of CBG, respectively, was accomplished employing the high-resolution mass spectrometry (HRMS) technique, and subsequently confirmed by comparison with the same compounds obtained by chemical synthesis. Complete spectroscopic characterization (NMR, FT-IR, UV, and HRMS) of both impurities is reported in the present work. The method was validated in terms of linearity, which was assessed in the range 0.01–1.00 μg/mL, sensitivity, selectivity, intra- and inter-day accuracy and precision, and short-term stability, which all satisfied the acceptance criteria of the ICH guidelines. Application of the method to the analysis of four commercial CBG samples highlighted a certain variability in the impurity profile that might be ascribed to the hemp variety of the starting plant material. With these new analytical standards in hand, it would be interesting to investigate their concentrations in different hemp varieties and expand the scope of a phytocannabinomics approach for a comprehensive profiling of this remarkable class of natural compounds.

Protein tyrosine O-sulfation is an important post-translational modification, as it has been correlated to inflammation, virus infection, and signal pathways. Nevertheless, methods for the characterization of protein sulfation by sulfopeptide enrichment are currently limited. In this Article, two standard compounds, representative of mono- and disulfated peptides, were used to compare the enrichment capabilities of five sorbent materials: two commercial weak anion-exchange mixed-mode sorbents (Strata X-AW and Oasis WAX) and three phosphopeptide enrichment materials based on affinity chromatography to either immobilized metals (IMAC) or metal oxides, i.e., Fe3+, TiO2, or Ti4+. The sulfopeptides were analyzed by ultrahigh-performance liquid chromatography (UHPLC) multiple-reaction monitoring analysis and were stable under all the tested experimental conditions. Recoveries of the enrichment step from spiked bovine serum albumin digests were >80% for the commercial Fe-IMAC kit and the Strata X-AW sorbent. Shotgun proteomics was used on the same samples to evaluate the selectivity, calculated as the number of coenriched peptides, and it was found to be better for the Fe-IMAC kit. Therefore, the Fe-IMAC protocol was embedded in a shotgun-proteomics workflow and applied to serum spiked with the sulfopeptides before protein dephosphorylation and digestion. The recovery of the entire analytical workflow was 20%, which was compatible with previous data on TiO2 phosphopeptide enrichment. Despite the potential, no sulfopeptide was confidently identified in serum digests by conventional shotgun proteomics, probably due to very low abundance of native sulfoproteins, poor ionization efficiency of sulfopeptides in the positive mode, and lack of unambiguous sulfopeptide identification by bioinformatics software. In this context, the use of negative-ionization mode with high-resolution mass spectrometry appeared promising to improve the sensibility and allow sulfopeptide identification in complex samples.

The chemical composition of Cannabis sativa L. has been extensively investigated for several years; nevertheless, a detailed lipidome characterization is completely lacking in the literature. To achieve this goal, an extraction and enrichment procedure was developed for the characterization of phospholipids and sulfolipids. Firstly, a study on the solid-liquid extraction was performed, to maximize the recovery of the considered lipids; the best procedure consisted of a simple extraction with a mixture of methanol and chloroform (1:1, v/v). The hemp extracts were analyzed by ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry and lipids were tentatively identified by Lipostar. To improve the number of identifications, an enrichment method, based on graphitized carbon black solid phase extraction, was evaluated to fractionate phospholipids and sulfolipids into separate eluates. Recovery and matrix effects of the procedure were determined on a mixture of standard lipids, containing representative compounds for each considered lipid class. The optimized method allowed the tentative identification of 189 lipids, including 51 phospholipids and 80 sulfolipids, in the first and second fractions, respectively. The detection of only 6 sulfolipids in the first fraction and 9 phospholipids in the second fraction proved the efficacy of the fractionation method, which also allowed the number of lipid identifications to be increased compared to the same procedure without enrichment, which scored 100 lipids. Finally, a semi-quantitative analysis permitted the hemp polar lipidome to be characterized. The results of this study allow knowledge of the hemp chemical composition to be improved with a detailed description of its phospho- and sulfolipid profiles. [Figure not available: see fulltext.]

Due to the rapidly increasing resistance to conventional antibiotics, antimicrobial peptides are emerging as promising novel drug candidates. In this study, peptide fragments were obtained from yellowfin tuna muscle by simulated gastrointestinal digestion, and their antimicrobial activity towards Gram-positive and Gram-negative bacteria was investigated. In particular, the antimicrobial activity of both medium- and short-sized peptides was investigated by using two dedicated approaches. Medium-sized peptides were purified by solid phase extraction on C18, while short peptides were purified thanks to a graphitized carbon black sorbent. For medium-sized peptide characterization, a peptidomic strategy based on shotgun proteomics analysis was employed, and identification was achieved by matching protein sequence database by homology, as yellowfin tuna is a non-model organism, leading to the identification of 403 peptides. As for short peptide sequences, an untargeted suspect screening approach was carried out by means of an inclusion list presenting the exact mass to charge ratios (m/z) values for all di-, tri- and tetrapeptides. In total, 572 short sequences were identified thanks to a customized workflow dedicated to short peptide analysis implemented on Compound Discoverer software.

Short peptides, namely di- tri- and tetra peptides, have been proven to play an important diagnostic role in several diseases. Therefore, the development of an analytical approach for their detection and identification is nowadays an important research goal. This paper describes an analytical procedure able to overcome the issues of short peptide isolation, clean-up and identification in plasma samples. Four different protocols were compared and tested to maximize both recovery and total number of identifications of short circulating plasma endogenous peptides. The purified peptides, coming from the four different tested protocols, were separated by zwitterionic hydrophilic liquid chromatography coupled to high-resolution mass spectrometry with the purpose of accomplishing an untargeted investigation based on suspect screening for short peptides in plasma. In particular, the use of Phree(TM) Phospholipid removal cartridge in combination with a purification step by solid phase extraction on a graphitized carbon black sorbent allowed the identification of the largest number of amino acid sequences (91 short peptides). The clean-up procedure allowed to tackle the issue of the low abundance of such peptides and their suppression during mass-spectrometric analysis. The results indicated that sample preparation is therefore fundamental for short peptide analysis in plasma samples.

Hemp and cannabis industry is undergoing a renewed interest due to legalization of marijuana (a topic that all countries are discussing, especially in recent years) and the growing importance of therapeutic properties of cannabinoids. Together with an increment in the production of hemp and recreational cannabis, there has been an increasing demand for accurate potency testing of products (i.e. quantification of main cannabinoids present in the plant in terms of weight percentage) prior commercialization. This translates in an urgent need of reliable analytical methods to characterize cannabis and hemp samples. Cannabis and hemp preparations are commercialized under various forms (e.g., flowers, oils, candies or even baked goods) usually containing a large number of often very similar compounds making their separation very challenging. Strictly connected to this, another emerging topic concerns the need for the developing of large scale separation techniques for the purification of cannabinoids from complex matrices and for the preparation of analytical-grade standards (including the chiral ones). This paper reviews the most recent achievements in both these aspects. Cutting-edge applications and novel opportunities in potency testing by high performance liquid chromatography (HPLC) with UV detection (which is becoming the golden standard, according to several pharmacopeias, for this kind of measurements) are discussed. The focus has been given to the very important topic of enantio-discrimination of chiral cannabinoids, for which supercritical fluid chromatography (SFC) appears to be particularly suitable. The last part of the work covers the purification of cannabinoids through preparative chromatography. In this regard, particular attention has been given to the most innovative multi-column techniques allowing for the continuous purification of target molecules. The most recent advancements and future challenges in this field are discussed.

The aim of this paper was the development of an analytical strategy for the production of purified bioactive peptides from cauliflower waste proteins, by testing two different extraction protocols and screening different enzymes for protein hydrolysis. The purification of peptides was carried out by multidimensional liquid chromatography employing reversed phase chromatography and hydrophilic interaction chromatography; the resulting fractions were tested for antihypertensive and antioxidative activities. The most active ones were characterized by nano-liquid chromatography-tandem mass spectrometry and identified by database search. The identified peptides were further mined by in-silico analysis using PeptideRanker to ascribe a bioactivity rank to each peptide. Thus, six potential ACE-inhibitory peptides were synthesized and validated checking their retention times and fragmentation patterns for consistency. Pure peptide standards were finally in-vitro tested for the specific bioactivity. In this way, three novel ACE-inhibitory peptides were successfully identified and validated from cauliflower waste hydrolysate, showing good IC50 values.

Recently, magnetic solid-phase extraction has gained interest because it presents various operational advantages over classical solid-phase extraction. Furthermore, magnetic nanoparticles are easy to prepare, and various materials can be used in their synthesis. In the literature, there are only few studies on the determination of mycoestrogens in milk, although their carryover in milk has occurred. In this work, we wanted to develop the first (to the best of our knowledge) magnetic solid-phase extraction protocol for six mycoestrogens from milk, followed by liquid chromatography and tandem mass spectrometry analysis. Magnetic graphitized carbon black was chosen as the adsorbent, as this carbonaceous material, which is very different from the most diffuse graphene and carbon nanotubes, had already shown selectivity towards estrogenic compounds in milk. The graphitized carbon black was decorated with Fe3O4, which was confirmed by the characterization analyses. A milk deproteinization step was avoided, using only a suitable dilution in phosphate buffer as sample pretreatment. The overall process efficiency ranged between 52 and 102%, whereas matrix effect considered as signal suppression was below 33% for all the analytes even at the lowest spiking level. The obtained method limits of quantification were below those of other published methods that employ classical solid-phase extraction protocols. This article is protected by copyright. All rights reserved

Donkey milk is a valuable product for the food industry due to its nutraceutical, nutritional, and functional properties. In this work, the endogenous peptides from donkey milk were investigated for their antioxidant and ACE-inhibitory activities, combining a two-dimensional peptide fractionation strategy with high-resolution mass spectrometry, bioinformatics analysis, and in vitro assays. After extraction, the endogenous peptides were fractionated twice, first by polymeric reversed phase and then by hydrophilic interaction chromatography. Fractions were screened for the investigated bioactivities and only the most active ones were finally analyzed by nanoRP-HPLC-MS/MS; this approach allowed to reduce the total number of possible bioactive sequences. Results were further mined by in silico analysis using PeptideRanker, BioPep, and PepBank, which provided a bioactivity score to the identified peptides and matched sequences to known bioactive peptides, in order to select candidates for chemical synthesis. Thus, five peptides were prepared and then compared to the natural occurring ones, checking their retention times and fragmentation patterns in donkey milk alone and in spiked donkey milk samples. Pure peptide standards were finally in vitro tested for the specific bioactivity. In this way, two novel endogenous antioxidant peptides, namely EWFTFLKEAGQGAKDMWR and GQGAKDMWR, and two ACE-inhibitory peptides, namely REWFTFLK and MPFLKSPIVPF, were successfully validated from donkey milk. Graphical Abstract Analytical workflow for purification and identification of bioactive peptides from donkey milk sample.

Estrogens, phytoestrogens, and mycoestrogens may enter into the surface waters from different sources, such as effluents of municipal wastewater treatment plants, industrial plants, and animal farms and runoff from agricultural areas. In this work, a multiresidue analytical method for the determination of 17 natural estrogenic compounds, including four steroid estrogens, six mycoestrogens, and seven phytoestrogens, in river water samples has been developed. (Fe3O4)-based magnetic nanoparticles coated by polydopamine (Fe3O4@pDA) were used for dispersive solid-phase extraction, and the final extract was analyzed by ultra-high performance liquid chromatography coupled with tandem mass spectrometry. The Fe3O4 magnetic nanoparticles were prepared by a co-precipitation procedure, coated by pDA, and characterized by scanning electron microscopy, infrared spectroscopy, and elemental analysis. The sample preparation method was optimized in terms of extraction recovery, matrix effect, selectivity, trueness, precision, method limits of detection, and method limits of quantification (MLOQs). For all the 17 analytes, recoveries were >70 % and matrix effects were below 30 % when 25 mL of river water sample was treated with 90 mg of Fe3O4@pDA nanoparticles. Selectivity was tested by spiking river water samples with 50 other compounds (mycotoxins, antibacterials, conjugated hormones, UV filters, alkylphenols, etc.), and only aflatoxins and some benzophenones showed recoveries >60 %. This method proved to be simple and robust and allowed the determination of natural estrogenic compounds belonging to different classes in surface waters with MLOQs ranging between 0.003 and 0.1 μg L(-1). Graphical Abstract Determination of natural estrogenic compounds in water by magnetic solid phase extraction followed by liquid chromatography-tandem mass spectrometry analysis.

Among the bioactive compounds present in extra-virgin olive oil, polar lipids and free fatty acids are minor compounds with well-known nutritional values and have been studied for traceability and adulteration investigations as well. In the present paper, the simultaneous characterization of polar lipids and free fatty acids in a pool of fifteen EVOO samples was achieved by means of reversed phase C18 analysis coupled to negative polarity high-resolution mass spectrometry. A total of 24 polar lipids, comprising 19 phospholipids and 5 sulfolipids, and 27 free fatty acids were tentatively identified, including several odd-chain and very long-chain fatty acids at trace levels. Moreover, a one-month study of lipid degradation on simulated storage conditions was carried out thanks to the set-up of a dedicated approach for degradation product analysis which was implemented of Compound Discoverer software. By virtue of the customized data processing workflow, more than forty compounds were tentatively identified, including compounds deriving from hydrolysis and oxidation reactions. Finally, by analysis of peak area trends, phosphoester hydrolyses of polar heads of phospholipids emerged as the fastest reactions, followed by glycerol ester hydrolyses and oxidative processes.

Short peptides are important compounds in a variety of fields, including food and nutraceutical applications, but also biomarker discovery, bioactive peptide discovery and peptide drug separation. Despite the importance of short peptides, they are currently less studied than other peptides because of the lack of dedicated methods for their characterization. The method described in this paper comprises a combination of strategies to tackle the main limitations in short peptide analysis. In particular, in this work an untargeted peptidomic approach based on ultrahigh-performance liquid chromatography coupled to high-resolution mass spectrometry was developed for the identification of short peptides in cow milk samples. After milk defatting and precipitation, the sample was purified by cotton-hydrophilic interaction liquid chromatography (HILIC) micro tip in order to avoid suppression phenomena due to contaminants present in milk, such as carbohydrates. The sample was then separated by means of two chromatographic columns, with a complementary selectivity mechanism, namely reversed-phase C18 column and porous graphitic carbon (PGC). By this approach, the method allowed the separation and characterization of di-, tri- and tetrapeptides. A total of 57 and 41 peptides were identified by using a C18 and a PGC column, respectively; in particular, 31 were exclusively identified by using the C18 column, 15 unique peptides were identified by using the PGC column, while 26 were in common between the two data sets, demonstrating that the two columns have a different selectivity mechanism. The results indicated that an integrated approach may be appropriate to improve the separation of different peptides and increase the number of identifications because of the wide range of polarity of short peptides. The method allowed the untargeted identification of short peptides in milk, a complex matrix chosen as a representative real sample for method application, and provides complementary information to that accessible by ordinary peptidomics. [Figure not available: see fulltext.].

Short peptide sequences represent emerging analytes in a variety of fields, including biomarker discovery, but also a well-known analytical challenge in complex matrices, due to the low abundance, extensive suppression during MS analysis, and lack of workflows, as they cannot be identified by ordinary peptidomics strategies and coverage is extremely limited by metabolomics as well. In this context, in this work, a solid phase extraction method was developed for the cleanup and enrichment of dipeptides, tripeptides, and tetrapeptides in urine using graphitized carbon black Carbograph 4 as the sorbent. The method was first developed on analytical standards spiked in urine, with recoveries in the range of 60-100%. Then the method was applied to urine samples from healthy volunteers. The enriched urine samples were analyzed by ultrahigh performance liquid chromatography (UHPLC) using an orthogonal strategy in which both a reversed phase (RP) C18 column and a zwitterionic hydrophilic interaction liquid chromatography (HILIC) column were used, for better coverage of peptide polarity and improved detection of peptides. High-resolution mass spectra were acquired in data-dependent mode using a suspect screening strategy with inclusion list; peptides were identified by a semiautomated workflow for feature extraction, candidate mass filtering, and MS/MS spectra comparison with in silico mass spectra. The complementarity of the orthogonal separation strategy was confirmed by peptide identification, resulting in 101 peptides identified from the RP runs and 111 peptides from the HILIC runs, with 60 common identifications. The method is applicable to both hydrophobic and hydrophilic peptides. Peptides were stable over 2 h after collection and protease inhibitors were not necessary, as no formation of artifacts was observed.

The combination of an efficient chromatographic separation with post-column addition of a supercharging agent was evaluated for the determination of small peptides. The procedure takes advantage of porous graphitic carbon (PGC) ability in retaining very polar and ionic molecules to overstep the poor retention of small peptides on conventional reversed phase (RP) columns. The method was developed specifically for the most hydrophilic di-, tri- and tetrapeptides, which are not identified in ordinary peptidomics experiments. In addition to retention mechanisms acting on conventional RP, the method exploited the charge induced interactions generated by the charges on the peptides with the polarizable surface of PGC. This results in efficient retention of very short and highly polar peptides using classical RP mobile phases. The effects of varying mobile phase composition (organic solvent and ion-pairing additives) as well as column temperature have been thoroughly investigated using short peptide standards. Under optimized conditions (water and acetonitrile/tetrahydrofuran 99:1 (v/v), both with 0.15% trifluoroacetic acid, as phase A and B, respectively, 0.5 mL min−1 flowrate at 50 °C) the effect of post-column addition of 3-nitrobenzylic alcohol was also investigated allowing effective coupling of the chromatographic system with high resolution mass spectrometry. Finally, an untargeted approach for peptide identification was pursued, based on precursor identification in database with all possible combinations of the 20 natural amino acids and fragmentation spectra matching to in silico generated spectra. The method was then applied to investigation of the short endogenous peptides in human serum from healthy individuals resulting in the identification of 30 short peptides.

Liquid chromatography coupled to low-resolution mass spectrometry (LRMS) has historically been a popular approach for compound quantitation. Recently, high-resolution mass spectrometry (HRMS) technical developments led to the introduction of new approaches for quantitative analysis. Whereas the performances of HRMS have been largely assessed for qualitative purposes, there are still questions about its suitability for quantitative analysis. Several papers on LRMS and HRMS comparison have been published; however, none of them was applied to polyphenol quantitation. In this work, a comparison between HRMS, operated in data-dependent acquisition mode, and LRMS, operated in selected-reaction-monitoring mode, was performed for polyphenol quantitation in wine. The two techniques were evaluated in terms of sensitivity, linearity range, matrix effect, and precision, showing the better performances of HRMS. The suitability of HRMS for quantitation purposes as well as qualitative screening makes HRMS the new technique of choice for both targeted and untargeted analysis.

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.

Microalgae have recently become a popular functional food due to their health benefits. Sulfolipids, a class of substances abundant in this matrix, have been reported to have interesting bioactivities, such as anti-carcinogenic activity. However, despite the potential interest in sulfolipids, a dedicated analytical method for their characterization is currently lacking but would significantly increase the coverage of sulfolipids with respect to the direct lipidomic analysis. To achieve this goal, in this work a procedure, based on graphitized carbon black solid phase extraction, was developed for clean-up and enrichment of sulfolipids (sulfoquinovosyldiacylglycerols and sulfoquinovosylmonoacylglycerols) and it was applied to spirulina (Arthrospira platensis) microalgae. A careful study of the solid phase extraction conditions was performed, first to maximize the recovery of reference standards, then to increase the total number of identified sulfolipids from the spirulina lipid extract. All samples were analysed by ultra-high performance liquid chromatography coupled to high resolution mass spectrometry and lipids were tentatively identified by Lipostar, for a reliable lipid structure assignment. The developed method was compared to the direct lipidomic analysis without enrichment, to establish the enrichment efficiency in terms of number of identifications. From the comparison, the enrichment procedure proved better and allowed the tentative identification of 199 sulfolipids, which is the largest number reported so far for the Arthrospira platensis species. The described method was validated in terms of precision, accuracy, recovery, limit of quantitation and detection for two sulfolipids. Finally, a relative lipid quantitation based on peak area was carried out on the microalgae sample, which indicated nine abundant sulfolipids as representing ca. 60% of sulfolipids in spirulina microalgae.

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.

Nowadays, there is an increasing attention for nutraceuticals and, in general, bioactive compounds naturally present in food. Indeed, the possibility of preserving human health and preventing disease (e.g., cardiovascular diseases, cancer etc.) by the intake of healthy food is attractive for both consumers and food industries. In turn, research in this field was also prompted significantly, with the aim of characterizing these bioactive compounds and ascribe to them a specific activity. The bioactive compounds can belong to several chemical classes. However, their chemical diversity and presence in complex matrices, such as food, make it challenging both their isolation and characterization. To tackle this issue, efficient separation systems are needed, which are mainly based on chromatography. In this context, this mini-review aims to provide the reader with an overview of the most relevant and recent approaches for the separation of the most common bioactive compounds in food, in particular polyphenols, phenols, carotenoids, and peptides, by liquid chromatography approaches.

Selenium is an essential micronutrient for humans. In food, selenium can be present in both inorganic and organic forms, the latter mainly being selenomethionine, Se-methyl-selenocysteine, and selenocystine. Selenoamino acid speciation rarely involves the chirality of selenoamino acids. In this work, a 5 cm long CHIROBIOTIC TAG chromatographic column was used for enantioresolution of selenoamino acids (d- and l-selenomethionine, Se-methyl-l-selenocysteine, d-, l- and meso-selenocystine); in the optimized conditions, the complete resolution of the analytes was achieved within 15 min by using a very polar aqueous mobile phase (gradient elution by methanol/acetonitrile/H2O, 45:45:10 (v/v/v) with 10 mmol L–1 of ammonium formate and 0.5% formic acid as the mobile phase A and acetonitrile/H2O, 20:80 (v/v) with 20 mmol L–1 of ammonium formate at apparent pH 4 as the mobile phase B). The affinity of the teicoplanin aglycone was further exploited to devise a preconcentration method for selenoamino acids in oils. I...

Protein post translational modifications currently represent one of the main challenges with proteomic analysis, due to the important biological role they play within cells. Protein phosphorylation is one of the most important, with several approaches developed for phosphopeptides enrichment and analysis, essential for comprehensive phosphoproteomic analysis. However, the development of new materials for phosphopeptides enrichment may overcome previous drawbacks and improve enrichment of such peptides. In this regard, new magnetic stationary phases based on polydopamine coating and Ti(4+) immobilization exploit the potential of IMAC enrichment and couple it with the versatility of magnetic solid phase extraction. In this work the use of such stationary phase was extended from the MALDI proof of concept stage with the development of an optimized method for phosphopeptides enrichment compatible with typical shotgun proteomics experimental workflows. Different loading and elution buffers were tested to improve phosphopeptides recovery and enrichment selectivity. Finally, the analysis of isolated peptides pointed out that polydopamine alone is an ideal support matrix for polar post translational modifications because it enables to reduce unspecific binding and preferentially binds hydrophilic peptides.

The proteome characterization of fish muscle tissues, together with the relative expression of each individual protein, provides knowledge on the biochemical response of the organisms and allows to assess the effect of different types of feeding, growth site and nutritional quality of the investigated species. This type of study is usually performed by gel-based proteomics approaches, however shotgun proteomics can serve as well, reducing analysis time and improving sample high-throughput. In this work, a shotgun proteomics method was thus developed and then applied to the characterization of gilthead sea bream edible muscle. The sarcoplasmic protein fraction was extracted, in-solution digested by trypsin and finally analyzed by nanoHPLC high resolution tandem mass spectrometry. Two different quantification strategies were also tested. One was based on chemical dimethyl labeling and the other one on label free quantification. A comparison between these two analytical workflows was performed, to evaluate their individual performance in the analysis of fish samples and assess the differences induced by farming practice on the final commercial product with respect to wild gilthead sea bream. Quantitative differences were detected, and the most relevant one regarded the common fish allergen parvalbumin, found overexpressed in farmed fish samples.

Natural estrogens are synthesized by mammals in different amounts depending on the developmental stage and pregnancy/lactation period, and they may pass into milk, where they are mostly present as glucuronated and sulfated forms. In modern dairy practices, about 75% of milk is produced from pregnant cows; therefore, the amount of hormones that may pass into milk could be of concern. While estrogen determination in milk has been investigated in depth, the individual determination of estrogens and their conjugated forms in dairy products has not been fully addressed. The aim of this work was to develop and assess a sensitive method, using the peculiar retention properties of graphitized carbon black, to extract natural free estrogens and their major conjugated metabolites, without any enzymatic cleavage, from yogurt, cheese, and butter. The free and conjugated estrogens were eluted in two distinct fractions from the solid-phase extraction cartridge and analyzed separately by ultra high performance liquid chromatography coupled to tandem mass spectrometry. Recoveries were higher than 80% for all the three sample typologies. The highest matrix effects were observed for butter, which was richest in lipid content, but was below 30%. A survey on some commercial dairy products suggests that production processes decreased estrogen content.

A novel method for the simultaneous determination of enniatins A, A1, B and B1 and beauvericin, both in human urine and plasma samples, was developed and validated. The method consisted of a simple and easy pretreatment, specific for each matrix, followed by solid phase extraction (SPE) and detection by high performance liquid chromatography-tandem mass spectrometry with an electrospray ion source. The optimized SPE method was performed on graphitized carbon black cartridges after suitable dilution of the extracts, which allowed high mycotoxin absolute recoveries (76%–103%) and the removal of the major interferences from the matrix. The method was extensively evaluated for plasma and urine samples separately, providing satisfactory results in terms of linearity (R2 of 0.991–0.999), process efficiency (&gt, 81%), trueness (recoveries between 85% and 120%), intra-day precision (relative standard deviation, RSD &lt, 18%), inter-day precision (RSD &lt, 21%) and method quantification limits (ranging between 20 ng·L−1 and 40 ng·L−1 in plasma and between 5 ng·L−1 and 20 ng·L−1 in urine). Finally, the highly sensitive validated method was applied to some urine and plasma samples from different donors.

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.

Interest in research into bioactive peptides (BPs) is growing because of their health-promoting ability. Several bioactivities have been ascribed to peptides, including antioxidant, antihypertensive and antimicrobial properties. As they can be produced from precursor proteins, the investigation of BPs in foods is becoming increasingly popular. For the same reason, production of BPs from by-products has also emerged as a possible means of reducing waste and recovering value-added compounds suitable for functional food production and supplements. Milk, meat and fish are the most investigated sources of BPs, but vegetable-derived peptides are also of interest. Vegetables are commonly consumed, and agro-industrial wastes constitute a cheap, large and lower environmental impact source of proteins. The use of advanced analytical techniques for separation and identification of peptides would greatly benefit the discovery of new BPs. In this context, this review provides an overview of the most recent applications in BP investigations for vegetable food and by-products. The most important issues regarding peptide isolation and separation, by single or multiple chromatographic techniques, are discussed. Additionally, problems connected with peptide identification in plants and non-model plants are discussed regarding the particular case of BP identification. Finally, the issue of peptide validation to confirm sequence and bioactivity is presented. Graphical representation of the analytical workflow needed for investigation of bioactive peptides and applied to vegetables and vegetable wastes Graphical Abstract.

A shotgun proteomics approach was used to characterize the quinoa seed proteome. To obtain comprehensive proteomic data from quinoa seeds three different precipitation procedures were employed: MeOH/CHCl3 /double-distilled H2 O, acetone either alone or with trichloroacetic acid; the isolated proteins were then in-solution digested and the resulting peptides were analyzed by nano-liquid chromatography coupled to tandem mass spectrometry. However, since quinoa is a nonmodel plant species, only a few protein sequences are included in the most widely known protein sequence databases. To improve the data reliability a UniProt subdatabase, containing only proteins of Caryophillales order, was used. A total of 352 proteins were identified and evaluated both from a qualitative and quantitative point of view. This combined approach is certainly useful to increase the final number of identifications, but no particular class of proteins was extracted and identified in spite of the different chemistries and the different precipitation protocols. However, with respect to the other two procedures, from the relative quantitative analysis, based on the number of spectral counts, the trichloroacetic acid/acetone protocol was the best procedure for sample handling and quantitative protein extraction. This study could pave the way to further high-throughput studies on Chenopodium Quinoa.

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.

UV filters, contained in sunscreens and other cosmetic products, as well as in some plastics and industrial products, are nowadays considered contaminants of emerging concern because their widespread and increasing use has lead to their presence in the environment. Furthermore, some UV filters are suspected to have endocrine disruption activity. In the present work, we developed an analytical method based on liquid chromatography with tandem mass spectrometry for the determination of UV filters in tap and lake waters. Sixteen UV filters were extracted from water samples by solid-phase extraction employing graphitized carbon black as adsorbent material. Handling 200 mL of water sample, satisfactory recoveries were obtained for almost all the analytes. The limits of detection and quantification of the method were comparable to those reported in other works, and ranged between 0.7-3.5 and 1.9-11.8 ng/L, respectively; however in our case the number of investigated compounds was larger. The major encountered problem in method development was to identify the background contamination sources and reduce their contribution. UV filters were not detected in tap water samples, whereas the analyses conducted on samples collected from three different lakes showed that the swimming areas are most subject to UV filter contamination.

As soon as nanomaterials, such as nanoparticles (NPs), are injected into a physiological environment a rich coating of biomolecules known as the "protein corona" rapidly covers them. This protein dress is the main factor, which affects the interaction of NPs with living systems. While the relationship between NP features and the biomolecule corona has been extensively investigated, whether and how changes in the physiological environment affect the NP-protein corona remains under-investigated. This is one of the most important steps in translating results in animal models to the clinic. Here we investigated thoroughly the biological identity of lipid NPs (size, charge, aggregation state and composition of the corona) after incubation with human plasma (HP) and mouse plasma (MP) by dynamic light scattering, micro-electrophoresis and nano-liquid chromatography tandem mass spectrometry (nanoLC/MS-MS). Specifically, we used two different liposomal formulations: the first one was made of polyethyleneglycol (PEG)-coated 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), while the second one was made of 30% of DOTAP, 50% of neutral saturated 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 20% cholesterol. The temporal evolution and complexity of the NP-protein corona was found to be strongly dependent on the biological environment. In MP, liposomes were more negatively charged, less enriched in opsonins and appreciably more enriched in apolipoproteins than their counterparts in HP. Collectively, our results suggest that the biological identities of NPs in mice and humans can be markedly different from each other. Relevance of results to in vivo applications is discussed.

In this work, liquid-liquid extraction (LLE) and solid phase extraction (SPE), of polyphenols from a VOO sample were optimised by a Plackett-Burman experimental design; then the two extraction techniques capabilities were compared. By using HPLC-DAD, the extraction ability of SPE with the diol phase and LLE were similar. The two methods were further evaluated with ultra HPLC-ESI QToF in negative ion mode by recoveries of standards and matched comparison of the peak area of 40 identified and 27 unidentified compounds. Conclusions indicate that LLE gives better recoveries for highly polar, non-polar, and some polyphenols suspected to contain a nitrogen atom, while for the others the two methods seem to be equally suitable. The presence of nitrogen-containing polyphenols was confirmed in positive ionisation mode in LLE extract, whereas in the SPE extract they were not present. One of them was tentatively identified as a compound containing tyrosine and methyl-decarboxymetyl-eleanoic acid moieties.

A sensitive, simple and rapid method for the simultaneous determination of 19 mycotoxins in biscuits (a dry matrix containing cereals and egg) has been developed using high performance liquid chromatography coupled to tandem mass spectrometry with electrospray source working in both positive and negative mode. Due to the matrix complexity and the high amount of contaminants, a solid phase extraction method using graphitized carbon black was optimized for an effective clean-up step. Accuracy was carried out in the selected matrix using blank samples spiked at three analyte concentrations. Recoveries between 63 and 107% and relative standard deviations lower than 12% were obtained. For all considered mycotoxin classes, i.e. thricotecenes A and B, zearalenone and its metabolites, fumonisins, ochratoxin A, enniatins and their structurally related beauvericin, the method was validated in terms of linearity, recovery, matrix effect, precision, limit of detection and limit of quantification. Matrix-matched calibration was used for quantification purposes, in order to compensate for matrix effect. The coefficients of determination obtained were in the range of 0.9927-1. The limits of quantification, ranging from 0.04μgkg(-1) for enniatin B1 to 80.2μgkg(-1) for nivalenol, were always lower than maximum permitted levels for every regulated mycotoxin by the current European legislation.

Zearalenone (ZEN) is a nonsteroidal estrogenic mycotoxin produced by various Fusarium species and commonly occurring in corn and other cereals. Even though its acute toxicity is low, still the estrogenic activity of ZEN and metabolites is a matter of concern. In this work, a new magnetic molecularly imprinted polymer (mMIP) for the selective extraction of ZEN from cereal flours is presented. The mMIP was synthesized previously using quercetin as dummy template, and here we wanted to test its applicability to complex food samples. Analyte determination was carried out by high-performance liquid chromatography coupled to tandem mass spectrometry. The selectivity of the mMIP and the main validation method parameters were assessed. In particular, even in samples as complex as cereals, matrix effect was negligible. Although the mMIP showed cross-selectivity towards both ZEN-related and quercetin-related compounds, nonetheless ZEN recovery was &gt, 95% for the two lower spiking levels, and the quantification limit was 0.14 ng g&minus, 1, i.e., ca. 500 times lower than the maximum limit fixed for most cereals by European law. Therefore, the material, also in comparison with a commercial sorbent, appears suitable for the application in food analysis, also to isolate ZEN at trace levels.

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.

This review describes the principles and instrumentation for the screening of asymmetric reactions by mass spectrometry. These techniques witnessed a significant advancement in the last few years. Although some of them are still at the proof-of-concept development stage, several applications might be foreseen in the field of combinatorial, high-throughput parallel catalyst screening.

Microparticles (MPs) are small fragments of apoptotic or activated cells that may contribute to pathological processes in many diseases. Platelet-derived MPs (PMPs) are the most abundant type of MPs in human blood. To characterize the proteins in PMPs we used a shotgun proteomics approach by nanoHPLC separation followed by MS analysis on an LTQ Orbitrap XL. PMPs were produced from isolated platelets stimulated with adenosine diphosphate (ADP). We developed an analytical platform constituted by two different steps: in the first one we used a standard shotgun strategy; in the second one, to improve low-molecular weight, low-abundance-proteins identification, the samples were fractionated using hydrogel nanoparticles, an enrichment system based on a mixed mechanism of dimensional exclusion and colorant affinity. This was chosen to tackle a common issue with shotgun approaches, in which the low-abundance proteins are not detected when surveys are on a broad scale. By means of the entire analytical platform, we identified 603 proteins, 243 of which were not previously identified. A simple and straightforward procedure for the study of PMPs was provided, producing a tool for further understanding their biological and pathological roles, and a baseline for future studies aimed at discovering biomarkers involved in several diseases.

Liquid chromatography coupled to high-resolution mass spectrometry is the technique of choice for the untargeted profiling of food matrices. Despite the high potential of high-resolution mass spectrometry, when dealing with complex mixtures, an efficient separation technique is also needed. The novel core–shell chromatographic columns packed with sub-2 μm sized particles are claimed to show very good resolution. However, the analytes retention can be significantly altered when working under ultra-high performance chromatographic conditions. In this work, an evaluation of four chromatographic systems, with either a single or two in-series KinetexTM C18 columns, either packed with 2.6 or 1.7 μm particles, is presented for the targeted analysis of a standard mixture and the untargeted analysis of a strawberry extract. An ultra-high performance chromatographic system coupled via an electrospray source to a hybrid quadrupole-Orbitrap mass spectrometer was used. From the extensive comparison, a surprising result was obtained, namely, that the system identifying the largest number of features was the one with two in-series connected columns with the larger particle size. The inconsistency among the theoretical assumptions and the applicative findings points out the importance of an extensive chromatographic evaluation for the comprehensive untargeted profiling of complex real samples. This article is protected by copyright. All rights reserved

Magnetic solid-phase extraction is one of the most promising new extraction methods for liquid samples before ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) analysis. Several types of materials, including carbonaceous ones, have been prepared for this purpose. In this paper, for the first time, the preparation, characterization, and sorption capability of Fe3O4-graphitized carbon black (mGCB) composite toward some compounds of environmental interest were investigated. The synthesized mGCB consisted of micrometric GCB particles with 55 m2 g−1 surface area bearing some carbonyl and hydroxyl functionalities and the surface partially decorated by Fe3O4 microparticles. The prepared mGCB was firstly tested as an adsorbent for the extraction from surface water of 50 pollutants, including estrogens, perfluoroalkyl compounds, UV filters, and quinolones. The material showed good affinity to many of the tested compounds, except carboxylates and glucoronates; however, some compounds were difficult to desorb. Ten UV filters belonging to the chemical classes of benzophenones and p-aminobenzoates were selected, and parameters were optimized for the extraction of these compounds from surface water before UHPLC–MS/MS determination. Then, the method was validated in terms of linearity, trueness, intra-laboratory precision, and detection and quantification limits. In summary, the method performance (trueness, expressed as analytical recovery, 85–114%; RSD 5–15%) appears suitable for the determination of the selected compounds at the level of 10–100 ng L−1, with detection limits in the range of 1–5 ng L−1. Finally, the new method was compared with a published one, based on conventional solid-phase extraction with GCB, showing similar performance in real sample analysis.

A new on-line multidimensional system for sequential trapping and individual elution and separation of peptides based on their molecular weight is described. By sequentially using two chemically different trapping columns, a polymethacrylate monolith and a packed C18 one, peptides from complex samples can be on-line trapped and divided into two fractions, containing respectively mainly medium-large peptides and smaller peptides. Then, by means of two switching valves working in parallel, the two fractions were individually separated by reversed phase chromatography. The whole gradient consisted of two subgradients, with the first one dedicated to the separation of smaller peptides and the second one to the separation of larger peptides. Such configuration allowed to identify up to 1476 proteins in a standard E. coli tryptic digest, with improved performance, increased average sequence coverage and reduced single unique peptide identifications compared to a conventional shotgun proteomics configuration comprising only the C18 trapping column and the analytical column.