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Simple Summary: In this study, a multiplexed strategy based on the combination of a nanoparticle-enabled blood test and serum levels of acute-phase proteins proved to be able to distinguish pancreatic cancer patients from healthy controls with a good and sex-dependent prediction ability. This study suggests a possible role of acute-phase proteins as pancreatic cancer biomarkers and paves the way for the development of multiplexed technologies for early cancer detection. The development of new tools for the early detection of pancreatic ductal adenocarcinoma (PDAC) represents an area of intense research. Recently, the concept has emerged that multiplexed detection of different signatures from a single biospecimen (e.g., saliva, blood, etc.) may exhibit better diagnostic capability than single biomarkers. In this work, we develop a multiplexed strategy for detecting PDAC by combining characterization of the nanoparticle (NP)-protein corona, i.e., the protein layer that surrounds NPs upon exposure to biological fluids and circulating levels of plasma proteins belonging to the acute phase protein (APPs) family. As a first step, we developed a nanoparticle-enabled blood (NEB) test that employed 600 nm graphene oxide (GO) nanosheets and human plasma (HP) (5% vol/vol) to produce 75 personalized protein coronas (25 from healthy subjects and 50 from PDAC patients). Isolation and characterization of protein corona patterns by 1-dimensional (1D) SDS-PAGE identified significant differences in the abundance of low-molecular-weight corona proteins (20–30 kDa) between healthy subjects and PDAC patients. Coupling the outcomes of the NEB test with the circulating levels of alpha 2 globulins, we detected PDAC with a global capacity of 83.3%. Notably, a version of the multiplexed detection strategy run on sex-disaggregated data provided substantially better classification accuracy for men (93.1% vs. 77.8%). Nanoliquid chromatography tandem mass spectrometry (nano-LC MS/MS) experiments allowed to correlate PDAC with an altered enrichment of Apolipoprotein A-I, Apolipoprotein D, Complement factor D, Alpha-1-antichymotrypsin and Alpha-1-antitrypsin in the personalized protein corona. Moreover, other significant changes in the protein corona of PDAC patients were found. Overall, the developed multiplexed strategy is a valid tool for PDAC detection and paves the way for the identification of new potential PDAC biomarkers. [ABSTRACT FROM AUTHOR]

Carbonaceous materials, namely graphitized carbon black (GCB) and porous graphitic carbon (PGC), are an exciting class of stationary phases attracting remarkable attention due to their peculiar characteristics of retention behavior for non-polar and polar analytes, becoming an alternative to the typical reversed-phase stationary phases. The present review is focused on the recent and critical analytical applications of GCB and PGC in liquid phase applications. A comprehensive overview of GCB in sample preparation was done, primarily solid-phase extraction and related techniques. Traditional analytes, such as pesticides and hormones, are reviewed along with new applications, especially short-chain peptides and polar metabolites, where this sorbent allowed for filling a gap in sample preparation from complex biological matrices. The second part of the review article describes the applications of porous graphitic carbon in liquid chromatography and supercritical fluid chromatography. The discussion covers the most relevant analyte classes, with an extensive description of the advantages and disadvantages of PGC over other chromatographic techniques. [Display omitted] • The difference between GCB and PGC are discussed. • The extraction and analysis application of GCB are summarized. • A recent application of GCB in short-chain peptidomics is discussed. • The chromatographic application of PGC are summarized. • The forecast and challenges in the use of carbonaceous material are illustrated. [ABSTRACT FROM AUTHOR]

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. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The structural and chromatographic characterization of two novel fluorinated mesoporous materials prepared by covalent reaction of 3-(pentafluorophenyl)propyldimethylchlorosilane and perfluorohexylethyltrichlorosilane with 2.5 μm fully porous silica particles is reported. The adsorbents were characterized by solid state 29Si, 13C, and 19F NMR spectroscopy, low-temperature nitrogen adsorption, elemental analysis (C and F), and various chromatographic measurements, including the determination of adsorption isotherms. The structure and abundance of the different organic surface species, as well as the different silanol types, were determined. In particular, the degree of so-called horizontal polymerization, that is, Si-O-Si bridging parallel to the silica surface due to the reaction, under 'quasi-dry' conditions, of trifunctional silanizing agents with the silica surface was quantified. Significant agreement was found between the information provided by solid-state NMR, elemental analysis, and excess isotherms regarding the amount of surface residual silanol groups, on the one hand, and the degree of surface functionalization, on the other. Finally, the kinetic performance of the fluorinated materials as separation media for applications in near-ultrahigh-performance liquid chromatography was evaluated. At reduced velocities of about 5.5 (ca. 600 bar backpressure at room temperature) with 3 mm diameter columns and toluene as test compound, reduced plate heights on the order of 2 were obtained on columns of both adsorbents. [ABSTRACT FROM AUTHOR]

Polyphenol concentrations were quantified by rapid resolution liquid chromatography/mass spectrometry in white table grape. The experimental vineyard was subjected to different kinds of water supply and supply rates. Samples from the same vineyards were also analysed after 6 weeks of storage in a refrigerator and after 6 weeks of delayed harvesting. Berry skins and seeds were analysed separately. A statistical treatment of the screening kind, namely the 2k full factorial design, was used for the interpretation of results. Storage, delayed harvesting and the different kinds of water supply appeared to be the variables most affecting grape polyphenol content. In some cases, results showed that polyphenol content diminished by more than 50% after 6 weeks if the grapes were stored in a refrigerator, or if a sprinkler was used for irrigation. [ABSTRACT FROM AUTHOR]

A label-free absolute quantitation method based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been developed. This methodology was applied to mouse heart in order to quantify cardiac troponin T (cTnT), which is considered to be a sensitive marker of heart damage. The cTnT was extracted, isolated by reversed-phase high-performance liquid chromatography, digested, and analyzed by MALDI-TOF MS. The MS-based quantitation was performed using matrix-matched calibration curves (due to a matrix effect) of two synthetic peptides, one cTnT-specific peptide and one internal standard peptide, respectively. Recoveries at three spiking levels ranged from 87–96%, with relative standard deviations of below 10%. The method detection limit and the method quantitation limit, expressed as the amount of cTnT for the amount of total sarcomeric protein extract, were 0.03 mg g−1 and 0.15 mg g−1, respectively. This method appears to be accurate and generally suitable for improving absolute protein quantitation. [ABSTRACT FROM AUTHOR]

The metabolic fate of the estrogenic mycotoxin zearalenone in rainbow trout is presently unknown. In this study, the tissue concentration of zearalenone and its principal metabolites (α-zearalenol and β-zearalenol) was determined. A known amount of zearalenone was administered as a single bolus to ten fish, and the biological tissue concentration was determined at various times following administration. The analytes were extracted from liver and muscular tissue using an on-line matrix solid-phase dispersion-solid-phase extraction sample preparation protocol, and their concentration determined by HPLC-Turboionspray-tandem mass spectrometry. The results showed that zearalenone is mainly metabolized into α-zearalenol in both liver and muscular tissues. The maximum concentrations of each analyte found in liver were 76.1, 211.2 and 63.7 ng/g respectively for zearalenone, α-zearalenol and β-zearalenol, while in muscular tissue they were 10.7, 8.2 and 6.5 ng/g. These values were reached after 2 h in liver tissue and 12 h in muscular tissue. Moreover the data obtained showed that the elimination rate in liver is quite fast since 48 h after the exposure less than 7% of the maximum concentration found is still present. In muscular tissue, however, about one-third of the maximum concentration found is still present after 48 h. [ABSTRACT FROM AUTHOR]

Describes a method on matrix solid-phase dispersion on-line with a solid-phase extraction cleanup process followed by liquid chromatography with mass spectrometry for the determination of 3 macrocyclic lactone mycotoxins in fish tissues. Limits of detection; Suppression of zearalenone.

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. [ABSTRACT FROM AUTHOR]

In this work, a heart-cut bidimensional achiral-chiral liquid chromatography method coupled to high-resolution mass spectrometry was developed for the separation of the main carboxylated phytocannabinoids, namely cannabidiolic acid (CBDA), tetrahydrocannabinolic acid (THCA), cannabichromenic acid (CBCA), and cannabicyclolic acid (CBLA), and decarboxylated derivatives, namely cannabidiol (CBD), Δ9-tetrahydrocannabinol (Δ9-THC), cannabichromene (CBC), and cannabicyclol (CBL), and the evaluation of their enantiomeric composition in extracts of different Cannabis sativa L. varieties. Optimal conditions for the chiral analysis of CBC- and CBL-type compounds were found with methanol and water (95:5, v/v , with 0.1% formic acid, 1.5 mL/min) on an amylose-based chiral stationary phase. These settings also allowed to evaluate the parameters responsible for CBC and CBCA racemization. [Display omitted] • Phytocannabinoids were analyzed by a heart-cut 2D achiral-chiral RP-HPLC-HRMS method. • CBCA enantiomers were separated for the first time in different cannabis varieties. • Enantiomerization studies were performed on isolated enantiomers of CBCA and CBC. • Temperature and UV light were investigated as responsible for erosion of enantiopurity. [ABSTRACT FROM AUTHOR]

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 C 18 , one column Kinetex Biphenyl, one column Kinetex core-shell XB-C 18 , two columns Kinetex core-shell XB-C 18 . 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• The (+) enantiomers of the main phytocannabinoids were synthesized. • Enantioseparation was developed and optimized by chiral RP-HPLC-DAD-HRMS/MS. • Carboxylated phytocannabinoids were isolated from the unheated cannabis extract. • Decarboxylated phytocannabinoids were isolated from the heated cannabis extract. • Enantiomeric composition of all main phytocannabinoids was evaluated. The evaluation of the chiral composition of phytocannabinoids in the cannabis plant is particularly important as the pharmacological effects of the (+) and (-) enantiomers of these compounds are completely different. Chromatographic attempts to assess the presence of the minor (+) enantiomers of the main phytocannabinoids, cannabidiolic acid (CBDA) and trans -Δ9-tetrahydrocannabinolic acid (trans -Δ9-THCA), were carried out on heated plant extracts for the determination of the corresponding decarboxylated species, cannabidiol (CBD) and trans -Δ9-tetrahydrocannabinol (trans -Δ9-THC), respectively. This process produces an altered phytocannabinoid composition with several new and unknown decomposition products. The present work reports for the first time the stereoselective synthesis of the pure (+) enantiomers of the main phytocannabinoids, trans -CBDA, trans -Δ9-THCA, trans -CBD and trans -Δ9-THC, and the development and optimization of an achiral-chiral liquid chromatography method coupled to UV and high-resolution mass spectrometry detection in reversed phase conditions (RP-HPLC-UV-HRMS) for the isolation of the single compounds and evaluation of their actual enantiomeric composition in plant. The isolation of the peaks with the achiral stationary phase ensured the absence of interferences that could potentially co-elute with the analytes of interest in the chiral analysis. The method applied to the Italian medicinal cannabis variety FM2 revealed no trace of the (+) enantiomers for all phytocannabinoids under investigation before and after decarboxylation, thus suggesting that the extraction procedure does not lead to an inversion of configuration. [ABSTRACT FROM AUTHOR]

Cannabidiolic acid (CBDA) and trans -Δ9-tetrahydrocannabinolic acid (trans -Δ9-THCA) are known to be the major phytocannabinoids in Cannabis sativa L., along with their decarboxylated derivatives cannabidiol (CBD) and trans -Δ9-tetrahydrocannabinol (trans -Δ9-THC). The cis isomer of Δ9-THC has been recently identified, characterized and quantified in several Cannabis sativa varieties, which had been heated (decarboxylated) before the analysis. Since decarboxylation alters the original phytocannabinoids composition of the plant, this work reports the identification and characterization of the carboxylated precursor cis -Δ9-THCA. The compound was also synthesized and used as analytical standard for the development and validation of a liquid chromatography coupled to high resolution mass spectrometry-based method for its quantification in ten Cannabis sativa L. samples from different chemotypes. The highest concentrations of cis -Δ9-THCA were found in CBD-rich varieties, lower levels were observed in cannabigerol (CBG)-rich varieties (chemotype IV) and in those varieties with a balanced level of both CBD and THC (chemotype III), while its levels were not detectable in cannabichromene (CBC)-rich varieties (chemotype VI). The presence of the cis isomer of THC and THCA raises the question on whether to include or not this species in the calculation of the total amount of THC to classify a cannabis variety as a drug-type or a fiber-type (hemp). [Display omitted] • cis -Δ9-Tetrahydrocannabinolic acid was identified for the first time in Cannabis sativa L. • An HPLC-HRMS method was developed and validated for the quantification of cis -Δ9-THCA. • All main phytocannabinoids were determined in ten cannabis samples. • cis -Δ9-THCA concentrations were higher in CBD-rich varieties. [ABSTRACT FROM AUTHOR]

The chromatographic retention mechanisms of two hydrophobic bonded phases, octadecyl ethyl-bridged organic/inorganic (BEH-C18) and straight-chain perfluorohexylpropyl silica (C6F13), have been investigated by using a homologous series of aliyl-benzenes and perfluoroalkyl acids as test compounds in a variety of acetonitrile/water mobile phases and at different temperatures. On both columns, polar compounds exhibited a characteristic U-shape retention behavior in function of acetonitrile amount in the eluent, whereas retention of neutral molecules decreased continuously, following an increase of organic modifier, over the entire mobile phase range. The dependence of perfluoromethylene selectivity upon eluent composition explains the typical reversed-phase behavior (decreasing in retention following an increase of acetonitrile in mobile phase) initially exhibited by perfluoroalkyl acids, but alone it cannot justify their increasing of retention at organic-rich mobile phases (approximately >90% v/v for acetonitrile with the C6F13 column and acetonitrile >80% v/v for the BEH-C18 one). It actually predicts an opposite trend, indicating thus the presence of mixed-mode retention mechanisms. Indeed it was found that, at organic-rich mobile phases, the transfer from the mobile to the stationary phase of the polar moiety of molecules drives retention. This finding has been correlated to the excess adsorption isotherm of acetonitrile/water binary mixtures and thus to the composition of the stationary phase. At organic-rich mobile phases, in feet, stationary phases are characterized by a positive excess of adsorbed water that creates an "environment" suitable to the transfer herein of polar groups. [ABSTRACT FROM AUTHOR]

Abstract: Mass spectrometry used in combination with a wide variety of separation methods is the principal methodology for proteomics. In bottom-up approach, proteins are cleaved with a specific proteolytic enzyme, followed by peptide separation and MS identification. In top-down approach intact proteins are introduced into the mass spectrometer. The ions generated by electrospray ionization are then subjected to gas-phase separation, fragmentation, fragment separation, and automated interpretation of mass spectrometric and chromatographic data yielding both the molecular weight of the intact protein and the protein fragmentation pattern. This approach requires high accuracy mass measurement analysers capable of separating the multi-charged isotopic cluster of proteins, such as hybrid ion trap-Fourier transform instruments (LTQ-FTICR, LTQ-Orbitrap). Front-end separation technologies tailored for proteins are of primary importance to implement top-down proteomics. This review intends to provide the state of art of protein chromatographic and electrophoretic separation methods suitable for MS coupling, and to illustrate both monodimensional and multidimensional approaches used for LC–MS top-down proteomics. In addition, some recent progresses in protein chromatography that may provide an alternative to those currently employed are also discussed. [Copyright &y& Elsevier]

Abstract: Polyphenol concentrations, including anthocyanidins, flavonols, flavan-3-ols and stilbenes, were quantified by liquid chromatography/mass spectrometry in two cultivars of red grapes for daily consumption, which were subjected to different kinds of water supply and nitrogen fertilisation rates. Samples from the same vineyards were also analysed after a 6week storage in a refrigerator and 6week delayed harvesting. Berry skins and seeds were analysed separately. In order to ascertain whether agronomical treatments, storage condition, and delayed harvesting could have an effect on the concentration of the polyphenol classes, a statistical treatment of the screening kind, namely 2 k full factorial design, was used for the interpretation of results. Storage, delayed harvesting and the different kinds of water supply appeared to be the variables mostly affecting grape polyphenol content. In some cases, results showed that polyphenol content diminished by more than 50% after 6weeks if the grapes were stored in a refrigerator. This approach also evidenced some interactions among the variables. [Copyright &y& Elsevier]

Abstract: A sensitive and reliable liquid chromatography/photoionization (APPI) tandem mass spectrometry method has been developed for determining nine selected mycotoxins in wheat and maize samples. The analytes were chosen on the basis of the mycotoxins under EU Commission Regulation (EC) No. 1881/2006, i.e., deoxynivalenol (DON), zearalenone (ZON), aflatoxins (AFs), and ochratoxin A (OTA), and considering the possibility of a near future regulation for T-2 and HT-2 toxins. Mycotoxins were extracted from samples by means of an one-step solvent extraction without any cleanup. The developed multi-mycotoxin method permits simultaneous, simple, and rapid determination of several co-existing toxins separated in a single chromatographic run, in which AFs, T-2 and HT-2 toxin are acquired in positive, while OTA, DON and ZON in negative mode. Although a moderate signal suppression was noticeable, matrix effect did not give significant differences at p =0.05. Then, calibration in standard solution were used for quantitation. Based on the EU Commission Decision 2002/657/EC, the method was in-house validated in terms of ruggedness, specificity, linearity, trueness, within-laboratory reproducibility, decision limit (CCα) and detection capability (CCβ). For all the analytes, the regression coefficient r ranged between 0.8752 (DON in wheat) and 0.9465 (ZON in maize), biases related to mean concentrations were from −13% to +12% of the nominal spiking level, and the overall within-laboratory reproducibility ranged 3–16%; finally, CCα values did not differ more than 20% and CCβ not more than 42% from their respective maximum limit. Method quantification limits ranged from 1/20 (AFG1) to 1/4 (AFG2 and OTA) the maximum limit established by European Union in the Commission Regulation (EC) No. 1881/2006 and its subsequent amendments. [Copyright &y& Elsevier]

Abstract: In this study, a magnetic bead-based platform amenable to high-throughput protein carbonic anhydrase II (CA II) capture is presented. The key steps in this approach involved immunoaffinity purification of the target protein from serum followed by on-bead digestion with trypsin to release a surrogate peptide. This tryptic peptide was quantified by liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS) operating in multiple reaction monitoring acquisition mode. Using a synthetic peptide standard and a structural analogue free-labeled internal standard, the resulting concentration was stoichiometrically converted to CA II serum concentration. The analytical steps, such as preparation of immunobeads, protein capture, proteolysis, and calibration, were optimized. The method was validated in terms of recovery (77%), reproducibility (relative standard deviation [RSD]<12%), and method detection limit (0.5pmolml−1). The developed method was applied to determining the CA II in eight healthy subjects, and the concentration measured was 27.3pmolml−1 (RSD=65%). [Copyright &y& Elsevier]

Abstract: A simple and rapid method able to determine residues of erythromycin A, tylosin and tilmicosin in whole eggs is presented here. The analytical protocol involves a one-step extraction followed by liquid chromatography (LC)–tandem mass spectrometry. Analytes were extracted from 1g of egg spiked with an internal standard (josamycin) with acetonitrile. In terms of accuracy, matrix effect and ion signal stability, no extract cleanup was found to be necessary. After partial solvent removal, the final extract was injected into the LC column. Extraction was effective, since absolute recovery of the analyte in egg at their maximum residue limit (MRL) level was 85–102%. Estimated limits of quantification (S/N=10) were 0.2–0.5ng/g. Based on the EU Commission Decision 2002/657/EC, the method was in-house validated in terms of ruggedness, specificity, linearity, within-laboratory reproducibility, decision limit (CCα) and detection capability (CCβ). The within-laboratory reproducibility, expressed as RSD (n =18 at the MRL levels), was not higher than 13%. After validation, a short study on EA depletion in eggs was conducted after administration of this drug to laying hens. [Copyright &y& Elsevier]

Abstract: We describe the development of a liquid chromatography with negative-ion atmospheric pressure photoionization tandem mass spectrometric (LC/NI-APPI/MS/MS) method for the simultaneous determination of tetrabromobisphenol A (TBBP-A) and five polybrominated diphenyl ethers (BDE-47, BDE-99, BDE-100, BDE-153 and BDE-154) in water. A mobile phase methanol/acetone/water was used, where acetone acts also as dopant. NI-APPI produced precursor ions corresponding to [M−H]− for TBBP-A, [M−Br+O]−, and [M−2Br+O]− for the BDE congeners studied. Each compound was quantified operating in multiple reaction monitoring mode. Linearity was observed in the range 0.025–10ng injected for all compounds. Coefficients of determination R 2 ranged from 0.9934 to 0.9982. BDEs were poorly retained by solid-phase extraction (SPE) from river water and sewage treatment plant effluent, thus liquid–liquid extraction (LLE) by n-hexane should be used for these samples. The recoveries of TBBP-A and PBDEs from tap water (SPE), river water and industrial wastewater (LLE) were in the range of 81–88%, 78–92%, and 43–99%, respectively, with relative standard deviations below 17%. The limits of detection, based on signal-to-noise ratio of 3, ranged from 0.004 to 0.1ng injected, and method quantification limits were 0.2–3.3ngL−1 but BDE47 (20.3ngL−1). Only TBBP-A was found in a treated industrial sewage at 4ngL−1, while BDE-99 and BDE-100 were detected on suspended solids. [Copyright &y& Elsevier]

Abstract: A simple and rapid method able to determine residues of seven quinolone antibacterials in whole eggs is presented here. This method is based on the matrix solid-phase dispersion technique with hot water as extractant followed by liquid chromatography–tandem mass spectrometry. After depositing 1.5g of an egg sample containing the analytes and the analyte surrogate (norfloxacin) on sand (crystobalite), this material was packed into an extraction cell. Quinolones were extracted by flowing 6mL of water acidified with 50mmol/L formic acid through the cell heated at 100°C. After pH adjustment and filtration of the extract, 100μL of it was injected into the LC column. MS data acquisition was performed in the multiple reaction monitoring mode, selecting two precursor ion to product ion transitions for each target compound. Hot water appeared an efficient extracting medium, since absolute recoveries of the analyte in egg at the level of 20ng/g were 89–103%. Estimated limits of quantification (S/N=10) were 0.2–0.6ng/g. Based on the EU Commission Decision 2002/657/EC, the method was validated in terms of ruggedness, specificity, linearity, within-laboratory reproducibility, decision limit (CCα and detection capability (CCβ). Depending on the particular analyte, CCαs ranged between 0.41 and 2.6ng/g, while CCβs were 0.64–3.7ng/g. The method was linear in the 3–30ng/g range, with typical R 2 values higher than 0.97. The within-laboratory reproducibility (n =21) at 6ng/g level was in the 9.0–12% range. After validation, a depletion study of enrofloxacin and one of its metabolites, i.e. ciprofloxacin, in eggs was conducted. [Copyright &y& Elsevier]

Abstract: A rapid, specific and sensitive procedure for determining residues of eight widespread used quinolone antimicrobials in bovine milk is presented. The method is based on the matrix solid-phase dispersion technique with hot water as extractant followed by LC/MS/MS. The entire sample treatment did not take more than 40min. Hot water appeared to be an efficient extracting medium, since absolute recoveries of the analytes in milk were 77–90%. The method proved to be robust as matrix effects did not affect significantly the accuracy of the method, as evidenced by analyzing six different batches of milk. Using norfloxacin as surrogate analyte, the accuracy of the method at three different spike levels of the analytes in milk was 93–110% with RSDs not larger than 10%. On the basis of a S/N of 10, estimated LOQs of this method range from 0.3 to 1.5ng/ml, well below the tolerance levels of quinolones in milk set by the European Union. [Copyright &y& Elsevier]

Abstract: A sensitive and reliable liquid chromatography–tandem mass spectrometric with electrospray ionization method for determining aflatoxins in hazelnuts has been developed. Three different extraction techniques, such as homogenization, ultrasonic extraction, and matrix solid phase dispersion have been tested and compared in terms of recovery, matrix effect, accuracy and precision. Ultrasound extraction was the most performing sample preparation method. Absolute recoveries for analytes and I.S. ranged from 93 to 101%. Accuracy and precision were calculated using matrix matched calibration, and ranged 91–102% and 2–11%, respectively. CCα and CCβ for aflatoxin B1 (EU limit=2μg/kg) were 2.15 and 2.33μg/kg, respectively. A ruggedness test performed on three other matrices demonstrated that sonication time was critical and a matrix matched calibration must be constructed for every sort of matrix. [Copyright &y& Elsevier]

Abstract: A confirmatory method for fusariotoxin analysis in maize meal, based on liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS), was developed, and compared with a previously published screening method, based on the same technique. By eluting selectively from a Carbograph-4 clean-up cartridge trichothecenes, fumonisins and macrocyclic lactones, and optimizing LC–MS/MS conditions for every chemical class, a sensitive and reliable determination was performed. Method quantification limits for confirmatory and screening methods were in the range 0.001–0.019mg/kg and 0.003–0.125mg/kg, respectively. Maize samples collected from four different hybrids grown in five experimental field trials were analyzed with both screening and confirmatory procedures. In most of the samples, fumonisin B1–3 were revealed with a concentration above 2mg/kg. Zearalenone was found at a higher level than 0.5mg/kg in three samples, and nine samples were found positive for this toxin only with the confirmation method, being contaminated at levels below 0.008mg/kg. Among trichothecenes B only deoxynivalenol was found twice at a concentration over 1mg/kg, whereas fusarenon X was never revealed. Trichothecenes A were present at a concentration lower than 0.015mg/kg. [Copyright &y& Elsevier]

Abstract: A solid phase extraction-high performance liquid chromatography–tandem mass spectrometry based analytical method suitable for simultaneous analysis of benzidine, 3,3′-dichlorobenzidine, mono-, di-, and tri-chloroanilines has been developed. Normal phase separation by liquid chromatography was performed using a cyano propyl methyl silica column, and atmospheric pressure photoionization was employed as interface with mass spectrometer. The developed method was evaluated in terms of limit of detection, accuracy, and precision. The quantification limit for all the compounds ranged between 7 and 112ngL−1, while recovery for all the compounds was higher than 94%. The method was tested by analyzing different industrial wastes, showing residual contamination by most of the analytes. [Copyright &y& Elsevier]

Abstract: A new method for determining aflatoxin M1 (AFM1) in cheese by liquid chromatography–tandem mass spectrometry has been developed. Two methodologies were compared for sample extraction. The first one involves sample extraction with dichloromethane for hard, aged cheese or acetone for fresh cheese and includes a preliminary matrix solid-phase dispersion–extraction step before solid-phase extraction (SPE) clean-up by a Carbograph-4 cartridge. The second method uses a water/methanol solution (90:10, v/v) extraction at 150°C before clean-up. The average recoveries of AFM1 from samples spiked at levels of 0.25–0.45μg/kg, were 81–92% and the precision (RSD) ranged from 3 to 7% with the first method, whilst the average recoveries were 79–84%, and RSD ranged from 7 to 15% for the second method. Due to different matrix effect, the quantification limits were 0.019–0.025μg/kg in the first case and 0.048–0.143μg/kg in the second one, depending on cheese typology. [Copyright &y& Elsevier]

Abstract: Anatoxin-a (AN) is a powerful neurotoxin that can be produced by cyanobacteria in eutrophic freshwaters. Consequently, AN can contaminate lakes, rivers and basins destined for drinking water and aquaculture. Two simple, specific and sensitive procedures for determining AN in lake water and fish muscle tissue are presented. Both analytical protocols are based on liquid-chromatography (LC)–tandem mass spectrometry (MS) with electrospray ionization. MS data were acquired in the multi reaction monitoring mode by selecting four precursor to product ion transitions. After filtration, AN in lake water was analyzed by directly injecting 0.5ml of the aqueous sample in the LC column. Analysis of AN in fish muscle tissue involved the matrix solid-phase dispersion technique. The analyte was extracted from tissue by 4ml of water acidified to pH 2 and heated at 80°C. After acidification and filtration, 0.2ml of the aqueous extract was injected in the LC column. Analyte recovery ranged between 71 and 79% and was not substantially affected by both the analyte concentration and the type of fish. Phenylalanine is an essential amino acid invariably present in any animal tissue. Like AN, this amino acid produces a pseudo molecular ion at m/z 166, it has a very similar fragmentation pattern and LC retention time. This method is able to prevent identifying phenylalanine for AN as the latter compound is eluted more than 1min before the former one and the two compounds have remarkably different relative ion signal intensities. On the basis of a signal-to-noise ratio of 10, limits of quantification of AN in water and fish fillet were estimated to be 13ng/l and 0.5ng/g, respectively. [Copyright &y& Elsevier]

Abstract: A liquid chromatography/electrospray (ESI)–tandem mass spectrometric method for the measurement of aflatoxin M1 (AFM1) in milk is described. Milk sample after protein precipitation with acetone was cleaned-up with a Carbograph-4 cartridge. Performances of the ESI source were compared with those of the atmospheric pressure photoionization source (APPI). Although a method quantification limit (MQL) of 6ng/kg could be achieved operating with APPI source with respect to an MQL of 12ng/kg with ESI, all the other performances being similar, then ESI was preferred as being more robust and widespread at present. [Copyright &y& Elsevier]

[Display omitted] • Optimization of Oasis HLB and GCB SPE sorbents for water contaminant extraction. • Comparable extraction recoveries observed for the 2 SPE sorbents (both used) • Untargeted analysis of river water samples by SPE and LC-high resolution (HR)MS. • Tentative identification of 241 compounds (mainly drugs) by match in HRMS database. • Different contamination levels depending on sample collection point of Rome municipality. 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 and 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. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • UHPLC-high resolution MS method developed for analysis of underivatized policosanols. • LiI was used in mobile phase for ionization and MS detection of native policosanols. • Targeted method was validated for C24, C26, C27, C28, and C30 policosanols in hemp. • C20-C33 policosanols were identified in hemp wax by untargeted analysis. [ABSTRACT FROM AUTHOR]

• Review of MS methods for characterization of phytocannabinoids in cannabis plant. • Description of targeted quantitative methods, mostly using GC-MS or LC-MS. • Description of untargeted analysis approaches of phytocannabinoids. • Untargeted analysis for discovery of new phytocannabinoids. • Untargeted analysis and chemometrics for improved cannabis classification. 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. [ABSTRACT FROM AUTHOR]

Industrial hemp (Cannabis sativa L.) represents an important plant, used for a variety of uses including pharmaceutical and nutraceutical purposes. As such, a detailed characterization of the composition of this plant could help future research to further exploit the beneficial effects of hemp compounds on the human health. Among the many compounds of hemp, fatty acids represent an interesting class of minor components, which has been overlooked so far. In this work, an untargeted approach based on liquid-chromatography coupled to a high-resolution mass spectrometry and a dedicated structure-based workflow for raw data interpretation was employed for the characterization of fatty acids from hemp inflorescences. A simple method, without any chemical derivatization, was developed for extraction and characterization of fatty acids leading to the tentative identification of 39 fatty acid species in the five hemp samples. A quantitative analysis on the untargeted data was initially performed, using peak areas as surrogate of analyte abundance for relative quantitation. Five fatty acids resulted the most abundant in all hemp samples, with ca. 90% of the total peak area. For these compounds a targeted quantitative method was validated, indicating that the most abundant ones were linolenic acid (1.39–7.95 mg g-1) and linoleic acid (1.04–7.87 mg g-1), followed by palmitic acid (3.74–6.08 mg g-1), oleic acid (0.91–4.73 mg g-1) and stearic acid (0.64–2.25 mg g-1). [Display omitted] • Minor constituents fatty acids were studied in five industrial hemp inflorescences. • Direct analysis without derivatization was used for method development. • 39 fatty acids identified by untargeted UHPLC-high resolution MS analysis. • Composition was studied by relative quantitation and targeted quantitative analysis. • Linoleic, linolenic, palmitic, oleic and stearic were the most abundant fatty acids. [ABSTRACT FROM AUTHOR]

• Development of an efficient clean-up strategy for short peptides in plasma. • Comparison of 4 different clean-up protocols (advantages and disadvantages). • Untargeted peptidomic approach for short peptide identification in plasma. • Identification of 91 short peptides, the largest number never identified before. • Development of a rapid and simple method for clinical applications. 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™ 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. [ABSTRACT FROM AUTHOR]