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The accidental ingestion of leaves or cut branches of oleander (Nerium oleander) often results in severe poisoning in cattle. This natural shrub contains oleandrin, a highly toxic cardiac glycoside, which may prove fatal even at very low amounts. For this reason, a highly sensitive ultra-high performance liquid chromatography-tandem mass spectrometry method was developed and validated for in vivo oleandrin determination in bovine blood and serum samples or in bovine liver and heart for post-mortem diagnosis. The method was highly selective as the mass spectrometer worked in selected reaction monitoring mode using the first five most intense m/z transitions of oleandrin. The limits of detection and quantitation were 0.11 and 0.36 ng mL(-1), respectively, whereas the method detection and quantitation limit values were in the range of 0.006-0.051 ng g(-1) and 0.018-0.154 ng g(-1), respectively. The chromatographic run was only 5 min and the time employed for the sample pretreatment (25-30 min) is useful to avoid the presence of matrix effects. All the recovery values were within 62.9-80.5%. The method was successfully used to analyse incurred samples from an accidentally poisoned cow and is expected to be applicable to forensic purposes

The paper deals with the development of an online UHPLC-MS/MS method for the identification and determination of 25 Endocrine Disrupting Chemicals (EDCs) belonging to different chemical classes (perfluorochemicals, bisphenols, alkylphenols, phthalates, and parabens). The study regards the possible EDC migration from different medical devices of diverse materials used in the routine hospitals for blood sampling or for the parenteral nutrition therapies. In order to evaluate the release of EDCs, the equipment used for blood sampling (syringes, butterfly, cannula-needle, microcapillary tube) was put in contact with a physiological solution at a prefixed time. As regards the migration tests carried out on the medical devices used in the parenteral nutrition therapy (infusion tubes, venous catheters), the catheters were undergone to internal contact with parenteral solution and external contact with physiological one, whereas the infusion tubes were subjected only to internal migration test with parenteral solution. The results demonstrated the released of some ECDs (such as parabens at the concentration of few pg mL−1, diethylphthalate and dibutylphthlate at ng mL−1, level), when using physiological solution as the leaching agent. In addition, the presence of a PFOS structural isomer was found at 29.0 ng mL-1: this compound was released in part from the infusion tubes and mostly from the venous catheters, particularly from silicone ones. Using a more lipophilic solution, such as parenteral preparations, a greater amount of EDCs was released, among which also the diethylhexylphthalate that was quantified in all the investigated devices (without highlighting particular trends or migration from PVC devices), but its concentration remained always lower than imposed maximum threshold limit on medical device of 0.1% w/w. The releases from the infusion tubes of the same materials (polyethylene) from different manufacturers are very comparable, whereas the catheter of silicone releases greater amount of EDCs than that of polyurethane.

Background Based on the medical history and laboratory analytical tests, a patient presenting symptoms compatible with Chronic Fatigue Syndrome was suspected of metal intoxication; therefore, a chelating therapy was attempted. In parallel, the profile of elemental excretion in urine was determined. Methods Chelation therapy by CaNa2EDTA was administered every two weeks and urine samples were routinely collected for 17 months. The samples were mineralized with HNO3 69 % and analyzed by Inductively-Coupled Plasma – Mass Spectrometry. Data were processed by multivariate statistical methods. Results Most of the toxic elements showed a peak of excretion in 12−24 h after EDTA administration, which returned to basal level by 24−36 h after the treatment. Yet, the excretion of some trace elements persisted in the urine collected 26 h after the treatment. Conclusions The analysis of excreted metals following the CaNa2EDTA infusion allowed to monitor dynamically the chelation therapy. The chelation therapy was effective in mobilizing and eliminating the principal heavy metals present from the body. However, since such clearance almost vanished 24 h after the treatment, a protocol with more frequent and low-dose administrations is advisable to improve the metal excretion.

Irinotecan (CPT-11) is a water-soluble anticancer drug widely used to treat several types of cancer. Even if the metabolites of CPT-11 are well-known and investigated, only limited information is available in the literature about the formation of photo-degradation products that can naturally originate from sunlight irradiation when the drug is released in aqueous systems. CTP-11 solutions at 10.0 mg L−1 were irradiated by simulated sunlight. The intensity of the drug decreased by 90% after 7.5 days of irradiation and no significant reduction of absorbance values was observed after 13 days. A sensitive UHPLC-MS/MS method was developed employing a hybrid triple quadrupole/linear ion trap mass spectrometer, that is able to work in data-dependent acquisition mode and to obtain information about the compounds formed during the photoirradiation. Moreover, a selected reaction monitoring method was built using the MS/MS fragmentation pattern of the compounds previously investigated. The method was validated considering LOD, LOQ, linearity, precision, selectivity, recovery and matrix effect. LOD and LOQ values were 0.02 and 0.05 ng mL−1, respectively, whereas MDL and MQL values in real water samples (river water, groundwater, well water, and wastewater) were lower than 0.05 and 0.2 ng mL−1, respectively. Eight photodegradation products were identified, among which five for the first time. Based on the MS and MS/MS fragmentation, the chemical structures of the degradation products were proposed. Hydrolysis experiments were carried out on the same solutions preserved in the dark, but no formation of other species was highlighted. The method was applied to several real samples: CPT-11 was detected and quantified only in a hospital effluent sample at the concentration of 0.41 ± 0.2 ng mL−1 together with the occurrence of PDP3. The outcomes of this study may be useful for updating the pollutant screening in water samples.

The characterization of 72 Italian honey samples from 8 botanical varieties was carried out by a comprehensive approach exploiting data fusion of IR, NIR and Raman spectroscopies, Proton Transfer Reaction - Time of Flight - Mass Spectrometry (PTR-MS) and electronic nose. High-, mid- and low-level data fusion approaches were tested to verify if the combination of several analytical sources can improve the classification ability of honeys from different botanical origins. Classification was performed on the fused data by Partial Least Squares - Discriminant Analysis; a strict validation protocol was used to estimate the predictive performances of the models. The best results were obtained with high-level data fusion combining Raman and NIR spectroscopy and PTR-MS, with classification performances better than those obtained on single analytical sources (accuracy of 99% and 100% on test and training samples respectively). The combination of just three analytical sources assures a limited time of analysis.

In this review, we give an overview of the actual proteomic approaches used in the study of cancer cells secretome. In particular, we describe the proteomic strategies to decipher cancer cell secretome initially focusing on the different aspects of sample preparation. We examine the issues related to the presence of low abundant proteins, the analysis of secreted proteins in the conditioned media with or without the removal of fetal bovine serum and strategies developed to reduce intracellular protein contamination. As regards the identification and quantification of secreted proteins, we described the different proteomic approaches used, i.e. gel-based, MS-based (label-based and label-free), and the antibody and array-based methods, together with some of the most recent applications in the field of cancer research. Moreover, we describe the bioinformatics tools developed for the in silico validation and characterization of cancer cells secretome. We also discuss the most important available tools for protein annotation and for prediction of classical and non-classical secreted proteins. In summary in this review advances, concerns and challenges in the field of cancer secretome analysis are discussed.

Selected ion monitoring (SIM) is an acquisition mass spectrometry (MS) scan mode used for monitoring one or a restricted number of ions. Unlike full scan acquisition, the MS analyzer during the SIM acquisition can spend more time for the selected ion, avoiding to acquire other ions that are not of interest for the analyst. This article takes into account the advantages and disadvantage of the SIM MS technique, considering that its use is generally coupled with separation techniques such as gas chromatography or high-performance liquid chromatography. An overview about sample introduction, ionization sources, and mass analyzers (both at low- and high-resolution) used during SIM experiments is here given. However, the increasing request of tandem mass spectrometry instrumentation is supplanting little by little the use of SIM acquisition in place of selected reaction monitoring (SRM), which is the evolution of SIM acquisition with tandem MS. A comparison between high-resolution SIM MS and SRM MS acquisition is also taken into account. At last, new trends of HR MS acquisitions are considered, in which generally the potentiality of data independent acquisition and of retrospective analysis can be utilized at the expense of the use of SIM experiments.

This article deals with the identification of the photo-degradation products of 4-aminocarminic acid potentially present in commercial beverages. Sixteen beverages of different composition but all containing the E120 dye were previously analysed by ultra-high-performance liquid chromatography (UHPLC) coupled with quadrupole-time of flight mass spectrometry to identify the common degradation products of the E120 dye. Since it is plausible to find unauthorised 4-aminocarminic acid in beverages which report generic E120 dye on the label, retrospective analysis was employed here not only to search for the possible presence of 4-aminocarminic acid but also to investigate the potential formation of photo-degradation products derived from this compound. For this purpose, a statistical approach based on Student's t-test was used to compare the degraded beverages containing 4-aminocarminic acid with all the others. Five degradation products were identified and their structures were elucidated on the basis of the high-accuracy and high-resolution of mass and mass/mass spectra. The toxicity of the degradation products was evaluated through the Ames Salmonella/microsome mutagenicity assay. No evidence of mutagenicity was obtained for the beverages subjected or not to irradiation, whereas a toxic effect of the 4-aminocarminic acid standard solution already at 100.0 µg l(-1) was found. This leads, once again, to the conclusion that the toxicity study must be carried out on the beverages in order to take into account of all the possible masking/protection interactions among the ingredients.

The study investigates the sunlight photodegradation process of carminic acid, a natural red colourant used in beverages. For this purpose, both carminic acid aqueous standard solutions and sixteen different commercial beverages, ten containing carminic acid and six containing E120 dye, were subjected to photoirradiation. The results show different patterns of degradation, not only between the standard solutions and the beverages, but also from beverage to beverage. Due to the different beverage recipes, unpredictable reactions take place between the dye and the other ingredients. To identify the dye degradation products in a very complex scenario, a methodology was used, based on the combined use of principal component analysis with discriminant analysis and ultra-high-performance liquid chromatography coupled with tandem high resolution mass spectrometry. The methodology is unaffected by beverage composition and allows the degradation products of carminic acid dye to be identified for each beverage. © 2014 Elsevier Ltd. All rights reserved.

The industrial processes of starch cationization are widely used and the workers must protect their health from the potential presence of toxic species, such as 2,3-epoxypropyltrimethylammonium chloride (ETA or Quab 151®) or 3-chloro-2-hydroxypropyltrimethylammonium chloride (CTA or Quab 188®) used during the industrial processes. In this study, a sensitive and fast chromatographic method for the identification and determination of these toxic species in samples placed in fixed air sampling positions near to the reactor of starch processing has been developed and fully validated. The method is based on hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry detection. The limits of detection are 3.0 ng mL−1 and 1.0 ng mL−1 for ETA and CTA, respectively, and the RSD% of the intraday (n = 5) and the interday (n = 35) precision of concentration determination are lower than 3.4% and 6.1%, respectively. The solid–liquid extraction procedure employed here is very simple (sonication) and fast (5 min) and allows obtaining reliable recovery values greater than 91.0%. No evidence of statistically significant matrix effects (at a 95% of confidence level) during the mass spectrometry analysis has been found. The method has been successfully applied to filter samples used in routine controls for the safety of workers involved in these type of industrial processes.

Head space (HS) solid phase microextraction (SPME) followed by gas chromatography with mass spectrometry detection (GC-MS) is the most widespread technique to study the volatile profile of honey samples. In this paper, the experimental SPME conditions were optimized by a multivariate strategy. Both sensitivity and repeatability were optimized by experimental design techniques considering three factors: extraction temperature (from 50°C to 70°C), time of exposition of the fiber (from 20 min to 60 min), and amount of salt added (from 0 to 27.50%). Each experiment was evaluated by Principal Component Analysis (PCA) that allows to take into consideration all the analytes at the same time, preserving the information about their different characteristics. Optimal extraction conditions were identified independently for signal intensity (extraction temperature: 70°C; extraction time: 60 min; salt percentage: 27.50% w/w) and repeatability (extraction temperature: 50°C; extraction time: 60 min; salt percentage: 27.50% w/w) and a final global compromise (extraction temperature: 70°C; extraction time: 60 min; salt percentage: 27.50% w/w) was also reached. Considerations about the choice of the best internal standards were also drawn. The whole optimized procedure was than applied to the analysis of a multiflower honey sample and more than 100 compounds were identified.

Proteins and small molecules from ancient objects and cultural heritage can provide key information and contribute to study the context of objects and artists. However, all present-day protocols and strategies for the analysis of ancient samples are often invasive and require microsampling. Here, we present a new method for the noninvasive analysis of proteins and small molecules: the technique uses a special ethyl-vinyl acetate film functionalized with strong cation/anion exchange and C8 resins, for interacting with both proteins and small molecules present on the surface of the objects, followed by LC-MS/MS analysis. The new method was fully validated for the determination of both proteins and small molecules on several types of supports, showing excellent analytical performances such as, for example, R2 of the calibration curve of 0.98 and 0.99 for proteins and small molecules, low but very repeatable recoveries, particularly adequate for investigations on precious ancient samples that must not be altered by the analytical procedure. ESEM images and LED multispectral imaging confirmed that no damages or alterations occurred onto the support surfaces and no residues were left from the extractive film. Finally, the new method was applied for the characterization of the binders of a historical fresco of the XVI century from the Flemish painter Paul Brill and of a recently discovered fresco from Isidoro Bianchi (XVII century). Moreover the method was employed for the identification of the colorant used by Pietro Gallo (XIV century) on a wood panel. The method here reported can be easily applied to any other research on ancient precious objects and cultural heritage, since it does not require microsampling and the proteins/small molecules extraction can be performed directly in situ, leaving the object unchanged and intact. (Figure Presented)

A metaproteomic approach, based on liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis, was followed to map the major bacterial metabolic functions associated with the rhizosphere of metal-tolerant and metal hyperaccumulator plants, growing in a serpentine soil naturally contaminated by heavy metals such as Ni, Co and Cr. In particular, an “in-house” bacterial protein database was built based on the genera recognised by 16S rDNA profiling, then it was used for protein identification from LC-MS data. The combination of the information arising from three different extraction protocols, applied to each soil sample, permitted the identification of almost 800 proteins, corresponding to functions assigned to proper Gene Ontology categories. Mainly proteins involved in response to stimulus or in transport of metals and nutrients revealed variability of bacteria responses to microenvironment conditions. As for taxonomy, Phyllobacterium, Microbacterium oxidans, Pseudomonas oryzihabitans, Stenotrophomonas rhizophila and Bacillus methylotrophicus bacterial species were more represented in the rhizosphere samples of the metal-tolerant Biscutella laevigata and of the Ni hyperaccumulator Noccaea caerulescens with respect to bulk soil. Combining 16S rRNA gene-based sequencing and metaproteomic analysis, we get insights into microbial community functions and their interaction with plants colonising the stressful environment of serpentine soils.

Pterocarpus marsupium is a well-known plant due to its healing properties, in particular, the use of its aqueous extract is able to reduce blood sugar levels and blood triglyceride concentrations. Although this plant has already been widely studied, a complete characterization of its aqueous extract has not been reported. The present study deals with the characterization of the aqueous extract of P. marsupium in order to obtain a full fingerprint of the volatile and nonvolatile constituents. The volatile constituents were identified by CG-MS, whereas the nonvolatile fraction was characterized by UHPLC-MS/MS using a nontarget approach. Several compounds were identified, in particular, polyphenolic species belonging to the class of proanthocyanidins. Cytotoxicity tests were carried out on four different cancer cell lines and three different non-tumoral cell lines. Preliminary results indicate a selective cytotoxicity of the aqueous extract towards the cancer cells. The potential cytotoxicity due to the presence of metals in the aqueous extract was ruled out by testing an aqueous mixture of the metals at the same concentration found in the P. marsupium extract.

In higher plants, photosystem II (PSII) is a multi-subunit pigment-protein complex embedded in the thylakoid membranes of chloroplasts, where it is present mostly in dimeric form within the grana. Its light-harvesting antenna system, LHCII, is composed of trimeric and monomeric complexes, which can associate in variable number with the dimeric PSII core complex in order to form different types of PSII-LHCII supercomplexes. Moreover, PSII-LHCII supercomplexes can laterally associate within the thylakoid membrane plane, thus forming higher molecular mass complexes, termed PSII-LHCII megacomplexes (Boekema et al. 1999a, in Biochemistry 38:2233–2239; Boekema et al. 1999b, in Eur J Biochem 266:444–452). In this study, pure PSII-LHCII megacomplexes were directly isolated from stacked pea thylakoid membranes by a rapid single-step solubilization, using the detergent n-dodecyl-α-d-maltoside, followed by sucrose gradient ultracentrifugation. The megacomplexes were subjected to biochemical and structural analyses. Transmission electron microscopy on negatively stained samples, followed by single-particle analyses, revealed a novel form of PSII-LHCII megacomplexes, as compared to previous studies (Boekema et al.1999a, in Biochemistry 38:2233–2239; Boekema et al. 1999b, in Eur J Biochem 266:444–452), consisting of two PSII-LHCII supercomplexes sitting side-by-side in the membrane plane, sandwiched together with a second copy. This second copy of the megacomplex is most likely derived from the opposite membrane of a granal stack. Two predominant forms of intact sandwiched megacomplexes were observed and termed, according to (Dekker and Boekema 2005 Biochim Biophys Acta 1706:12–39), as (C2S2)4 and (C2S2 + C2S2M2)2 megacomplexes. By applying a gel-based proteomic approach, the protein composition of the isolated megacomplexes was fully characterized. In summary, the new structural forms of isolated megacomplexes and the related modeling performed provide novel insights into how PSII-LHCII supercomplexes may bind to each other, not only in the membrane plane, but also between granal stacks within the chloroplast.

The secretome is the complex set of molecules secreted by cells; these molecules play a key role in cell signaling, communication and migration. Secretomics has been already used to discover new potential diagnostic biomarkers and therapeutic agents and to elucidate key autocrine pathways. Malignant mesothelioma (MMe), an extremely aggressive tumor, is characterized by a long latency period (20–30 years), a poor prognosis, and limited effective therapies. MMe has a highly secretory cell type, and the factors released by cells may act in an autocrine or paracrine fashion on tumor and stroma, where they may modulate the extracellular environment. The aim of this work is to characterize the secretome of two MMe cell lines, MM98 and REN, in comparison with a mesothelial cell line Met5A, in order to evaluate differences and similarities of these two different MMe cancer model systems, and to identify potential biomarkers. We performed quantitative shotgun proteomics using SWATH-MS technology and we identified a total of 421 proteins, 112 expressed in the secretome of REN cells, 208 expressed in the secretome of MM98 cells and 189 secreted by mesothelial cells; 25 proteins are shared by the two mesothelioma cell lines. Biological significance This study characterizes the secretome signature of the REN and MM98 cell lines, confirming the availability of a cell-culture based model in order to describe the cell-specific properties, and to provide a list of putative cancer biomarkers. This work constitutes the first qualitative and quantitative proteomic approach performed on MMe secretome. Moreover, since the data were acquired in SWATH-MS acquisition mode, they can be successively re-mined without performing a new analysis of the sample, which is extremely useful for retrospective analyses. The overall aim was to identify novel tumor-derived protein biomarkers with the potential to be applied for early diagnosis, prognosis, therapy prediction and/or disease monitoring of MMe.

An on-line solid phase extraction (SPE) ultra high performance liquid chromatography tandem mass spectrometry method has been developed for the simultaneous identification and determination of thirteen polycyclic aromatic hydrocarbons (PAHs) and twelve aldehydes (derivatized with 2,4-dinitrophenylhydrazine). The chromatographic conditions have been optimized to obtain the maximum of sensitivity and resolution taking into account the different retention interactions and the different ionization conditions of PAHs and derivatized aldehydes. LOD values ranging from 0.028 to 0.768 μg L −1 for PAHs and from 0.002 to 0.125 μg L −1 for aldehydes were obtained. The resolution permitted the separation of four couples of PAH isomers. Sample pre-treatment and SPE were optimized in order to apply the whole methodology to the analysis of different food matrices as salmon, frankfurter, steak, and pork chop, subjected to different cooking modes (smoked, grilled, cooked in oil or in butter). Particular attention was devoted to the evaluation of matrix effect that was significantly reduced through the on-line SPE treatment. For each food matrix the method detection limits, the method quantitation limits, and the recovery R were evaluated. R was shown not to depend on analyte concentration in the explored concentration range (LOQ – 50.000 μg L −1 ): the average R percent ranges from 70.6% to 120.0%.

A reversed-phase high-performance liquid chromatography (HPLC) method was developed for the simultaneous determination in food of biogenic amines and their precursor amino acids after a precolumn derivatization with dansyl chloride. The chromatographic conditions, selected to be suitable for mass spectrometry detection, were optimized through experimental design and artificial neural networks. The HPLC-UV method was validated by comparing the separation results with those obtained through a HPLC method, working under the same chromatographic conditions but employing mass spectrometry detection. The HPLC-UV method was then applied to the analysis of different food samples, namely, cheese, clams, salami, and beer. For all of the matrices, recoveries (relative standard deviation always 92% were obtained. The results are discussed as a function of the total biogenic amine content and of the concentration ratio between amines and precursor amino acids

When mass spectrometry is not combined to separation techniques, the evaluation of signal and noise in a complex mass spectrum is not trivial. The tuning of the spectrometer based only on the increase of the signal of a selected number of m/z values does not ensure the achievement of the best experimental conditions: signal could improve and noise could increase as well. The scope of this work is the development of a function separating signal and noise (for evaluating the S/N) from complex mass spectra for potential use as target function for the automatic tuning of the instrument. Two different methods were applied: the first is based on the separation of a pool of m/z values attributable to the signal from the m/z values due to the noise, while the second is based on the application of principal component analysis to separate the signal (present in the significant components) from the noise (present in the residuals). The comparison of the two methods was carried out by the evaluation of the stability of the signal and the target functions obtained, and the evaluation of the variation of the target functions as a function of concentration.

A new ultra high performance liquid chromatography coupled with tandem mass spectrometry method for a fast and sensitive determination of eight polyphenols (hydroxytyrosol, catechin, epicatechin, epigallocatechin gallate, oleuropein, quercetin, rutin, tyrosol) and panthotenic acid in extra-virgin olive oil was developed. The method does not require long sample pre-treatment and presents the lowest limit of detection and limit of quantitation values present in literature. Inter- and intra-day variability, linear dynamic range of the calibration curve, recovery and matrix effect were also determined and investigated. The method was applied to several oil samples of different type and origin. Given its accuracy, precision and rapidity, the method is characterized by an interestingly high throughput, reliability, and sensitivity.

This paper reports the study of the photodegradation reactions that tricyclazole can naturally undergo, under the action of sunlight, in aqueous solutions of standard tricyclazole and of the commercial BEAM(TM) formulation. The analyses are carried out by ultra-high performance liquid chromatography technique coupled with high-resolution tandem mass spectrometry. Analysis of both tricyclazole and BEAM(TM) water solutions undergone to hydrolysis does not evidence new chromatographic peaks with respect to the not treated solutions. On the contrary, analysis of the same samples subjected to sunlight irradiation shows a decreased intensity of tricyclazole signal and the presence of new chromatographic peaks. Two photodegradation products of tricyclazole have been identified, one of which has been also quantified, being the commercial standard available. The pattern is similar for the solutions of the standard fungicide and of the BEAM(TM) formulation. The results obtained from eco-toxicological tests show that toxicity of tricyclazole standard solutions is greater than that of the irradiated ones, whereas toxicity levels of all the BEAM(TM) solutions investigated (non-irradiated, irradiated, and hydrolyzed) are comparable and lower than those shown by tricyclazole standard solutions. Experiments performed in paddy water solution show that there is no difference in the degradation products formed.

This article deals with the photodegradation of sulforhodamine B, a dye widely used in nonpermanent tattoos. Degradation evidence was obtained from both aqueous and sweat-simulating solutions of the dye after 9 days of Solarbox irradiation. The identification of the degradation products was achieved using a nontarget approach. For this purpose, a micro liquid chromatography method coupled with tandem high-resolution mass spectrometry was developed. In addition, the chemical structures of five degradation products and two dye impurities were elucidated. The degradation products were the same for both types of solution, whereas the degradation rate of the dye in sweat-simulating solution was slightly faster than that in aqueous solution. The method was also applied to samples of tattooed pigskin subjected to irradiation, in order to better simulate the irradiation effects on the dye used on the skin. None of the degradation products found in the sulforhodamine B solutions were identified in the degraded tattooed pigskin samples, but a new signal at m/z 637.3051 (positive ionization) was found, and the structure of the corresponding molecule was elucidated. The mutagenicity of the photodegradation products was evaluated using a quantitative structure-activity relationship approach, which gave negative results for all the structures elucidated. Graphical Abstract Comparison between tattoed pigskin before and after photodegration process. Strategies for the identification of sulforhodamine B degradation products.

The study deals with the identification of the degradation products formed by simulated sunlight photoirradiation in a commercial beverage that contains Allura Red AC dye. An UHPLC-MS/MS method, that makes use of hybrid triple quadrupole/linear ion trap, was developed. In the identification step the software tool information dependent acquisition (IDA) was used to automatically obtain information about the species present and to build a multiple reaction monitoring (MRM) method with the MS/MS fragmentation pattern of the species considered. The results indicate that the identified degradation products are formed from side-reactions and/or interactions among the dye and other ingredients present in the beverage (ascorbic acid, citric acid, sucrose, aromas, strawberry juice, and extract of chamomile flowers). The presence of aromatic amine or amide functionalities in the chemical structures proposed for the degradation products might suggest potential hazards to consumer health.

The use of doping agents, once restricted to professional athletes, has nowadays become a problem of public health, since it also concerns young people and non-competing amateurs in different sports. The use is also diffused in social life for improving physical appearance and enhancing performance and even dietary supplements assumed to improve performance often contain anabolic steroids. While decades ago the so-called " classical doping agents" (like stimulants and narcotics) were used, to-day anabolic steroids are more widely diffused. Anabolic steroids are synthetic substances prepared by introducing modifications in the molecular structure of testosterone, the main natural androgenic anabolic steroid that forms in testes interstitial cells. The first report concerning the use of anabolic steroids by an athlete who searched for increased weight and power dates 1954. In 1974 the misuse of anabolic steroids in sports was banned by the International Olympic Committee and control tests were implemented in 1976 Montreal Olympic Games through radioimmunoassay analysis: the technique, however, only allows for unspecific detection of a limited number of exogenous steroids. Over the years, always new doping substances are synthesized and, as a consequence, the list of prohibited compounds is continuously updated and new suitable analytical methods for their detection and determination in biological matrices are continuously required. In doping control analysis the knowledge of steroid metabolism pathway in human body is of primary importance and the analytical methods must permit the simultaneous detection and determination not only of the forbidden precursor agents but also of their metabolites. In addition, the potential presence and amount in the biological samples of species that can interfere in the analysis should be evaluated. Also the several anabolic steroids, specifically designed to circumvent doping control, put on the market have been incorporated in the list of the prohibited substances of the World Anti-Doping Agency (WADA). In WADA list steroids figure in three main classes, namely anabolic steroids, corticosteroids and substances with anti-estrogenic properties. It must be strongly reminded that assumption of doping agents not only leads to athletes the possible failing of doping tests but causes important health risk and WADA prohibited list establishes criteria to highlight the alteration of the natural steroid profile caused by exogenous administration. Doping control analyses are generally performed in urine, a matrix that provides a prolonged detection time window, and less often in blood, serum, plasma, hair, saliva, and nails. To identify the chemical structures of anabolic steroids the use of mass spectrometry detection is very advantageous. Gas chromatography-mass spectrometry (GC-MS) techniques allowed for the development of comprehensive screening methods. GC-MS methods are sensitive and robust but present the disadvantages of time-consuming sample pretreatment, that is often based on hydrolysis and derivatisation reactions. Liquid chromatography-mass spectrometry (LC-MS) methods have been successfully used to identify and determinate steroids in different matrices, as well as to study their metabolisms. Nowadays, automatic rapid ultra high performance liquid chromatography (UHPLC) tandem mass spectrometry has become the technique of choice for steroid analysis. Due to its generally higher speed, sensitivity, reproducibility and specificity with respect to HPLC, it can be used to simultaneously separate and determinate multi component steroid mixtures. The technique is of huge interest to separate conjugates anabolic androgenic steroids, as it allows efficiency enhancement due to the small particle (sub-2. μm) column packing, which provides high peak capacity within analysis times even 5-10 fold shorter than conventional HPLC methods. Modern multiplex instruments can analyze thousands of samples per month so that, notwithstanding the generally high instrumental costs, the cost of the individual assay is affordable. In addition, the improved specificity and resolution offered by time-of-flight or quadrupole time-of-flight mass spectrometry allow their application in doping control analysis or in steroid profiling for accurate and sensitive full mass range acquisition. Aim of the present review is to consider, compare and discuss the applications of the UHPLC/MS methods present in literature for the identification and determination of forbidden steroids and their metabolites in human biological matrices

The broad group of biogenic amines includes polyamines and catecholamines, whose presence in human tissues and biological fluids can give important diagnostic information and act as marker of many pathologies. In particular, polyamines are involved in cancer cell growth while catecholamines act as neurotransmitters and hormones. Their simultaneous determination in biological tissues and fluids is therefore an important task. A high-performance liquid chromatography tandem mass spectrometry method is presented here for the simultaneous determination in urine of 16 biogenic amines: adrenaline (epinephrine), agmatine, cadaverine, dopamine, histamine, 3-methoxytyramine, noradrenaline (norepinephrine), norephedrine, octopamine, 2-phenylethylamine, putrescine, serotonin, spermidine, spermine, tryptamine, and tyramine. The method does not require any derivatization step. To guarantee the maximum of sensitivity, the mass spectrometer works in selected reaction monitoring mode, monitoring for each analyte the two most intensive transitions. Method validation includes the evaluation of limits of detection (that range from 0.3 to 6.6 μg L(-1)), limits of quantitation (that range from 1.0 to 21.9 μg L(-1)), linearity range (three orders of magnitude), recovery, intra- and inter-day precision on both concentration, and retention time. Recovery (R) is shown not to depend on the analyte concentration: the average R percent ranges from 72.9 to 100.0 %. Particular attention is devoted to the matrix effect and the correlated phenomena of ion enhancement or suppression in mass spectrometry detection.

To address the growing interest of consumers for information on the provenance of foodstuffs, the production chain of world renowned Moscato d’Asti white wine has been studied using the distribution of lanthanides as chemical markers. From soil to must, upon every stage of the chain, samples have been taken and analysed with ICP-MS in order to verify whether the original fingerprint of soil is maintained or not along the chain. Results of this traceability study show clearly that lanthanides fingerprint is kept unaltered in the passage soil-grapes-must, while fractionation occurs upon wine clarifying with bentonites. The second part of the work involves a study on 102 samples of Moscato d’Asti musts in order to verify how they reflect the features of the different geographical zones where they come from, and to build a basis to be able to identify possible adulterations performed by addition of foreign musts.

The study deals with the identification of the degradation products formed by simulated sunlight photoirradiation in a beverage that contains Allura Red AC (E129) dye. An UHPLC-MS/MS method that makes use of high resolution quadrupole-time-of-flight mass spectrometer, was developed. For the identification of the degradation products the software tool Information Dependent Acquisition (IDA) was used to automatically obtain information about the high resolution MS and MS/MS spectra of the species present. Accurate mass data were essential for the identification of the appropriate elemental composition: the elucidation of unknown chemical structures was obtained by using powerful software to mine the recorded chromatogram. The identified degradation products are formed from side-reactions and/or from the interactions taking place among the dye and other ingredients present in the beverage (i.e. ascorbic acid, citric acid, sucrose, aromas, strawberry juice and extract of chamomile flowers). The presence of aromatic amine or amide groups in the chemical structures of the degradation products suggests the potential hazard for the consumer health.

A rapid on-line solid phase extraction ultra high performance liquid chromatography tandem mass spectrometry method was developed for the identification and quantitation of nine perfluorinated compounds in matrices of environmental, biological and food interest. Pre-treatment, solid phase extraction, chromatographic and mass detection conditions were optimised, in order to apply the whole methodology to the analysis of different matrices. Particular attention was devoted to the evaluation of matrix effect and the correlated phenomena of ion enhancement or suppression in mass spectrometry detection. LOD and LOQ range from 3 to 15 ng L−1 and from 10 to 50 ng L−1, respectively. Method detection limits (MDLs) were also calculated for each kind of matrix. The recovery, evaluated for each analyte, does not depend on analyte concentration in the explored concentration range: average R ¯ % values are always greater than 82.9%. In the whole, the results obtained for samples of river waters, blood serum, blood plasma, and fish confirm the ubiquitous presence of perfluorinated compounds, as recently denounced by many sources.

Contamination of water resources is one of the major problems to be faced for environment preservation and sustainability. The monitoring of target compounds based on mass spectrometry and selected reaction monitoring mode is often insufficient to definitely assess the quality of surface water. Also potentially harmful non-target pollutants simultaneously present must be taken into account. Liquid chromatography coupled with tandem mass spectrometry is suitable to obtain complete information on water composition. Hybrid mass spectrometers such as triple quadrupole/linear ion trap, hybrid quadrupole/time-of-flight and linear ion trap/orbitrap analyzers should be used. Here, we review ultra-high performance liquid chromatography coupled with mass spectrometry methods developed for post-target and non-target screening analysis of water emerging contaminants, such as pesticides and their degradation products, pharmaceuticals and drug side-reaction products, surfactants and illicit drugs. The major points are the following: (1) the possibility of performing retrospective analysis only by high-resolution mass analyzer; (2) the compatibility of mass analyzer with ultra-high performance liquid chromatography; (3) the use of deconvolution software to detect unknowns; and (4) the limited availability of library database.

The study investigates the natural photodegradation pathway of mono-chloroanilines in river waters, with the aim to identify the predominant photoproducts formed. At this purpose a new sensitive on-line SPE HPLC–MS/MS method has been developed with LOQ values equal or lower than the legal threshold concentration levels allowed for mono-chloroanilines in waters. The degradation processes of o-, m- and p-chloroaniline have been investigated subjecting their solutions, prepared both in ultrapure and in river water, to sun light irradiation simulated by a solar box system. The SPE HPLC–MS/MS methodology allowed to evaluate the degradation kinetics, to identify the predominant photodegradation products and to propose the chemical structures. Two photoproducts (aniline and 3-aminophenol), for which standards are available, have also been quantified.