Comparison of chemometric strategies for potential exposure marker discovery and false-positive reduction in untargeted metabolomics: application to the serum analysis by LC-HRMS after intake of Vaccinium fruit supplements.

Untargeted liquid chromatographic-high-resolution mass spectrometric (LC-HRMS) metabolomics for potential exposure marker (PEM) discovery in nutrikinetic studies generates complex outputs. The correct selection of statistically significant PEMs is a crucial analytical step for understanding nutrition-health interactions. Hence, in this paper, different chemometric selection workflows for PEM discovery, using multivariate or univariate parametric or non-parametric data analyses, were comparatively tested and evaluated. The PEM selection protocols were applied to a small-sample-size untargeted LC-HRMS study of a longitudinal set of serum samples from 20 volunteers after a single intake of (poly)phenolic-rich Vaccinium myrtillus and Vaccinium corymbosum supplements. The non-parametric Games-Howell test identified a restricted group of significant features, thus minimizing the risk of false-positive retention. Among the forty-seven PEMs exhibiting a statistically significant postprandial kinetics, twelve were successfully annotated as purine pathway metabolites, benzoic and benzodiol metabolites, indole alkaloids, and organic and fatty acids, and five (i.e. octahydro-methyl-ß-carboline-dicarboxylic acid, tetrahydro-methyl-ß-carboline-dicarboxylic acid, citric acid, caprylic acid, and azelaic acid) were associated to Vaccinium berry consumption for the first time. The analysis of the area under the curve of the longitudinal dataset highlighted thirteen statistically significant PEMs discriminating the two interventions, including four intra-intervention relevant metabolites (i.e. abscisic acid glucuronide, catechol sulphate, methyl-catechol sulphate, and α-hydroxy-hippuric acid). Principal component analysis and sample classification through linear discriminant analysis performed on PEM maximum intensity confirmed the discriminating role of these PEMs.

Liquid chromatographic/electrospray ionization quadrupole/time of flight tandem mass spectrometric study of polyphenolic composition of different Vaccinium berry species and their comparative evaluation.

Ultra-high-performance liquid chromatography coupled with high-resolution quadrupole-time of flight mass spectrometry with both negative and positive ionization was used for comprehensively investigating the phenolic and polyphenolic compounds in berries from three spontaneous or cultivated Vaccinium species (i.e., Vaccinium myrtillus, Vaccinium uliginosum subsp. gaultherioides, and Vaccinium corymbosum). More than 200 analytes, among phenolic and polyphenolic compounds belonging to the classes of anthocyanins, monomeric and oligomeric flavonols, flavanols, dihydrochalcones, phenolic acids, together with other polyphenolic compounds of mixed structural characteristics, were identified. Some of the polyphenols herein investigated, such as anthocyanidin glucuronides and malvidin-feruloyl-hexosides in V. myrtillus, or anthocyanindin aldopentosides and coumaroyl-hexosides in V. uliginosum subsp. gaultherioides and a large number of proanthocyanidins with high molecular weight in all species, were described for the first time in these berries. Principal component analysis applied on original LC-TOF data, acquired in survey scan mode, successfully discriminated the three Vaccinium berry species investigated, on the basis of their polyphenolic composition, underlying one more time the fundamental role of mass spectrometry for food characterization.

Fully automated on-line solid phase extraction coupled to liquid chromatography-tandem mass spectrometry for the simultaneous analysis of alkylphenol polyethoxylates and their carboxylic and phenolic metabolites in wastewater samples.

Three different sorbents (i.e. endcapped octadecylsilane, octasilane and styrene-N-vinylpiperidinone co-polymer) were investigated in order to develop an on-line solid phase extraction-liquid chromatographic tandem mass spectrometric method (on-line SPE-LC-MS/MS) for the simultaneous analysis of alkylphenols polyethoxylate (AP(n)EOs, n = 1-8) and corresponding monocarboxylate (AP1ECs) and phenolic (APs) metabolites. The endcapped octadecylsilane was selected due to its full compatibility with a chromatographic approach, which allowed the elution of positively and negatively ionisable compounds in two distinct retention time windows, using a water-acetonitrile-tetrahydrofuran ternary gradient and a pellicular pentafluorophenyl column. On this SPE sorbent, the composition of the loading/clean-up solution was then optimized in order to achieve the best recoveries of target analytes. Under the best experimental conditions, the total analysis time per sample was 25 min and method detection limits (MDLs) were in the sub-nanograms per litre to nanograms per litre range (0.0081-1.0 ng L(-1)) for AP(n)EOs with n = 2-8, AP1ECs and APs, whereas for AP1EOs, an MDL of about 50 ng L(-1) was found. Using the mass-labelled compound spiking technique, the method performance was tested on inlet and outlet wastewater samples from three activated sludge treatment plants managing domestic and industrial sewages of the urban areas and the textile district of Prato and Bisenzio valley (Tuscany, Italy); in most cases, apparent recovery percentages approximately in the ranges of 50-110% and 80-120% were found for inlet and outlet samples, respectively. The on-line SPE-LC-MS/MS analysis of wastewater samples highlighted the presence of target analytes at concentrations ranging from few nanograms per litre to thousands nanograms per litre, depending on the compound and matrix analysed. AP2ECs were also tentatively identified in outlet samples.

Evaluation of different QuEChERS procedures for the recovery of selected drugs and herbicides from soil using LC coupled with UV and pulsed amperometry for their detection.

Seven quick, easy, cheap, effective, rugged and safe (QuEChERS)-based procedures, differing in both the extraction and clean-up steps, were investigated for the recovery of bentazone (BTZ), atrazine (ATZ), carbamazepine (CBZ), phenytoin (PNT) and its metabolite 5-(p-hydroxyphenyl-),5-phenylhydantoin (HPPH) from soil. Target analytes were chosen for their extensive use and/or occurrence in soil, as well as for their medium-high polarity characteristics (log K OW values in the range 0.88-2.80), which have been reported as a critical parameter for the recovery from soil with QuEChERS approaches. Liquid chromatography coupled with UV and pulsed amperometric (PA) detection at a glassy carbon electrode was used as instrumental technique. The recovery data obtained within each tested procedure were discussed for each compound investigated, highlighting different behaviour depending on the specific physicochemical characteristics of the analytes. The optimized QuEChERS conditions consisted of the extraction of analytes with CH3CN:H2O 70:30, 5 % CH3COOH, followed by a dispersive solid-phase extraction (d-SPE) clean-up step with C18 sorbent. This method, in which water is directly added to the soil together with acetonitrile and salts, allowed the rehydration step to be avoided, which can be as long as 30 min. Matrix effects were evaluated for both the detection techniques at different concentration levels, and they were below 24 % for both the detection technique used. The recoveries were evaluated at three concentration levels by a matrix-matched calibration and were in the ranges of 83-113 % (relative standard deviations (RSD) ≤ 14 %) and 88-109 % (RSD ≤ 11 %) for UV and PA detection, respectively, highlighting very good performances of the method, even for the more polar analytes. Method detection limits ranged from 4 µg/kg (BTZ) to 493 µg/kg (PNT) and from 4 µg/kg (HPPH) to 11 µg/kg (BTZ) for UV and PA detection, respectively. The method was finally compared with a microwave-assisted extraction procedure which provided less satisfactory extraction performances than the optimized QuEChERS procedure.

LC-MS/MS analysis of coccidiostats in meat supply chain safety.

The presence of coccidiostats in meat products represents an important topic because of the animal administration of these substances, authorized as feed additives for targeted species, in order to prevent and inhibit coccidiosis. Coccidiostats include both ionophores and synthetic molecules characterized by different chemical-physical properties such as polarity. Meat is a matrix characterized by many interfering compound groups, such as proteins, phospholipids, and fats. High-performance liquid chromatography (HPLC) coupled to mass spectrometry (MS) analysis allows the required selectivity and sensitivity for discriminating analytes and matrix interferences. For these reasons, an LC-MS/MS method for the analysis of coccidiostats in meat products was developed without SPE purification steps. The correct analyte quantification is allowed by matrix-matched calibration. The method validation was performed by the replicated analysis of spiked meat samples at two different concentration levels (limit of quantification-LOQ-and a 10 times LOQ) in order to evaluate method recovery and repeatability, plus spiked samples at higher concentrations up to 10,000 µg/kg. Moreover, the metrological approach was used for the calculation of method uncertainty. The application of the developed method to real samples evidenced the presence of some non-ionophores coccidiostats in the meat and liver of chicken and rabbit species. Although, the determined concentration was below the established MRLs, the monitoring of coccidiostats in the meat supply chain is confirmed as a good strategy in order to safeguard consumer health.

Incidence of Perfluoroalkyl Substances in Commercial Eggs and Their Impact on Consumer's Safety.

Eggs play an important role in a balanced diet; however, the European Food Safety Authority (EFSA) recognizes eggs as a major source of poly and per-fluoroalkyl substances (PFASs). In this study, the presence of PFASs was analysed in eggs produced by hens from Northern Italian regions, a PFASs-contaminated area. Sixty-five samples were analysed by high-performance liquid chromatography coupled with high-resolution mass spectrometry. The greatest presence of PFASs was found in eggs from Veneto and Emilia Romagna, and the most detected PFASs were perfluorobutanoic acid (PFBA) and perfluorooctanesulfonic acid (PFOS) (mean concentrations 0.30 ± 0.15 and 0.05 ± 0.00 ng g-1). Considering the most recent updates for the sum of the main four PFASs, the highest concentration found in the analysed samples was 0.05 ng g-1, well below the maximum limit set by the European Union. The PFAS intake evaluation confirmed that egg consumption does not represent a risk for Italian consumers.

Green and cost-effective synthesis of copper nanoparticles by extracts of non-edible and waste plant materials from Vaccinium species: Characterization and antimicrobial activity.

The aim of this work was the green synthesis of copper nanoparticles (Cu-NPs) using aqueous extracts of (i) bilberry (Vaccinium myrtillus L.) waste residues from the production of fruit juices and (ii) non-edible "false bilberry" fruits (Vaccinium uliginosum L. subsp. gaultherioides). Different cupric salts (CuCl2, Cu(CH3COO)2 and Cu(NO3)2) were used for the synthesis. The formation of stable nanoparticles (CuNPs) was assessed by transmission electron microscopy and the oxidation state of copper in these aggregates was followed by X-ray photoelectron spectroscopy. The polyphenol composition of the extracts was characterized, before and after the synthesis, using spectrophotometric methods (i.e. total soluble polyphenols and total monomeric anthocyanins) and high-performance liquid chromatography coupled with tandem mass spectrometry (i.e. individual anthocyanins). Polyphenol concentration in the extracts was found to decrease after the synthesis, indicating their active participation to the processes, which led to the formation of Cu-NPs. The antimicrobial activity of Cu-NPs, berry extracts, and cupric ion solutions were analysed by broth microdilution and time-kill assays, on prokaryotic and eukaryotic models. The antimicrobial activity of Cu-NPs, especially those derived from bilberry waste residues, appeared to be higher for both Gram-negative and Gram-positive bacteria, and for fungi, compared to the ones of its single components (cupric salts and berry extracts). Therefore, Cu-NPs from the green synthesis here proposed can be considered as a cost-effective sanitization tool with a wide spectrum of action.

Untargeted Metabolomics Analytical Strategy Based on Liquid Chromatography/Electrospray Ionization Linear Ion Trap Quadrupole/Orbitrap Mass Spectrometry for Discovering New Polyphenol Metabolites in Human Biofluids after Acute Ingestion of Vaccinium myrtillus Berry Supplement.

In this work, liquid chromatography, coupled with an electrospray ionization hybrid linear ion trap quadrupole/Orbitrap mass spectrometry, has been used to accurately identify polyphenol metabolites in human serum and urine after acute ingestion of a V. myrtillus berry supplement. The supplement was obtained by cryo-milling of bilberries, which were freeze-dried within 1 week after their harvesting, to maintain the berry native composition. Thirty-six derivatives of benzoic acids, hydroxyhippuric acids, cinnamic acids, phenylpropionic acids, phenylvaleric acids, phenylpentenoic acids and abscisic acid, together with two berry-native anthocyanins, one flavonol metabolite and two catechol derivatives were putatively identified in the investigated biofluids. The annotated compounds included 13 metabolites, among glucuronides and sulphates of phenylvaleric and phenylpentenoic acids, which have been identified for the first time in human biofluids after ingestion of V. myrtillus berries. It should be emphasized that the presence of phenylvaleric and phenylpentenoic acid derivatives is in agreement with their origin from fruit native flavanol monomers and oligomers, which are widely distributed in Vaccinium berries, but usually overlooked in metabolomics studies regarding bilberry. The identification of these compounds confirmed the key-role of untargeted metabolomics approach in the discovery of new metabolites which could be biologically active. Graphical Abstract.

Determination of Rare Earth Elements in multi-year high-resolution Arctic aerosol record by double focusing Inductively Coupled Plasma Mass Spectrometry with desolvation nebulizer inlet system.

An inductively coupled plasma sector field mass spectrometer (ICP-SFMS) was used to develop an analytical method for the fast determination of Na, Al, Sc, Ti, V, Cr, Mn, Fe, Ni, Cu, Zn, As, Y, Mo, Cd, Ba, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, and Pb in Arctic size-segregated aerosol samples (PM10), after microwave acidic digestion. The ICP-SFMS was coupled with a microflow nebulizer and a desolvation system for the sample introduction, which reduced the isobaric interferences due to oxides and the required volume of sample solutions, compared to the usual nebulization chamber methods. With its very low limit of detection, and taking into account the level of blanks, this method allowed the quantification of many metals in very low concentration. Particular attention was given to Rare Earth Elements (REEs - La to Lu). The efficiency in the extraction of REEs was proved to be acceptable, with recoveries over 83% obtained with a Certified Reference Material (AMiS 0356). The analytical method was then applied to particulate matter samples, collected at ground level in Ny Ålesund (Svalbard Islands, Norway), during spring and summer, from 2010 to 2015, with daily resolution and using a low-volume device. Thus, for the first time, a large atmospheric concentrations dataset of metals in Arctic particulate matter at high temporal resolution is presented. On the basis of differences in LREE/HREE ratio and Ce and Eu anomalies in spring and summer samples, basic information to distinguish local and long-range transported dust were achieved.

Applicability of the direct injection liquid chromatographic tandem mass spectrometric analytical approach to the sub-ngL-1 determination of perfluoro-alkyl acids in waste, surface, ground and drinking water samples.

The applicability of a direct injection UHPLC-MS/MS method for the analysis of several perfluoroalkyl acids (PFAAs) in a wide range of water matrices was investigated. The method is based on the direct injection of 100µL of centrifuged water sample, without any other sample treatment. Very good method detection limits (0.014-0.44ngL-1) and excellent intra and inter-day precision (RSD% values in the range 1.8-4.4% and 2.7-5.7%, respectively) were achieved, with a total analysis time of 20min per sample. A high number of samples - i.e. 8 drinking waters (DW), 12 ground waters (GW), 13 surface waters (SW), 8 influents and 11 effluents of wastewater treatment plants (WWTPIN and WWTPOUT) were processed and the extent of matrix effect (ME) was calculated, highlighting the strong prevalence of |ME| < 20%. The occurrence of |ME| > 50% was occasionally observed only for perfluorooctanesulphonic and perfluorodecanoic acids. Linear discriminant analysis highlighted the great contribution of the sample origin (i.e. DW, GW, SW, WWTPIN and WWTPOUT) to the ME. Partial least square regression (PLS) and leave-one-out cross-validation were performed in order to interpret and predict the signal suppression or enhancement phenomena as a function of physicochemical parameters of water samples (i.e. conductivity, hardness and chemical oxygen demand) and background chromatographic area. The PLS approach resulted only in an approximate screening, due to the low prediction power of the PLS models. However, for most analytes in most samples, the fitted and cross-validated values were such as to correctly distinguish between | ME | higher than 20% or below this limit. PFAAs in the aforementioned water samples were quantified by means of the standard addition method, highlighting their occurrence mainly in WWTP influents and effluents, at concentrations as high as one hundred of µgL-1.

Determination of phthalate diesters and monoesters in human milk and infant formula by fat extraction, size-exclusion chromatography clean-up and gas chromatography-mass spectrometry detection.

A sensitive and reliable analytical method was developed for the simultaneous determination of five phthalate diesters and corresponding monoesters in human milk samples and infant formulas. The method involved a liquid-liquid extraction with a CH2Cl2/CH3OH/NaCl 30% 2/1/0.5 (v/v/v) mixture, the clean-up of the extract by size-exclusion chromatography (swelling and elution solvent: cyclohexane/ethyl acetate 9/1v/v), the derivatization of monoesters by trimethylsilyl-diazomethane and instrumental analysis by gas chromatography coupled with mass spectrometry. Recovery was in the range of 83-115% and precision was found between 9% and 21%. For phthalate diesters, method detection limits (MDLs) ranged from hundreds of ng/kg to 4.2µg/kg on a fresh weight milk (f.w.) basis, depending on blank contribution evaluated in matrix. Lower MDLs (0.03-0.8µg/kg f.w.) were achieved for corresponding monoesters. The proposed method was applied to the determination of target compounds in nine human milk samples and four infant formulas, confirming their presence in all samples. However, a generally higher contamination was assessed in artificial milk than in breast milk samples.

Polyphenolic profiles and antioxidant and antiradical activity of Italian berries from Vaccinium myrtillus L. and Vaccinium uliginosum L. subsp. gaultherioides (Bigelow) S.B. Young.

Total soluble polyphenols (TSP), total monomeric anthocyanins (TMA), radical scavenging activity (RSA), ferric reducing antioxidant power (FRAP), and a number of anthocyanins, phenolic acids, coumarins, flavanols, dihydrochalcones and flavonols were investigated in Tuscan bilberry (i.e. Vaccinium myrtillus) and "false bilberry" (i.e. Vaccinium uliginosum subsp. gaultherioides Bigelow). V. myrtillus berries showed much higher TSP, TMA, RSA and FRAP values than V. uliginosum subsp. gaultherioides fruits. Moreover, very different profiles of individual phenolics were observed in the two species, being V. myrtillus mainly characterised by delphinidin and cyanidin glycosides, together with chlorogenic acid, and V. uliginosum subsp. gaultherioides dominated by malvidin derivatives and flavonols. Strong differences between the two species regarded also metabolites investigated herein for the first time, such as scopoletin, which was approximately two magnitude orders higher in V. uliginosum subsp. gaultherioides than in V. myrtillus berries. Very different abundances were also highlighted for cryptochlorogenic acid and quercetin-3-rhamnoside that were about ten-fold higher in bilberry than in "false bilberry". When the anthocyanin composition pattern of Tuscan "false bilberry" was compared to those elsewhere reported for V. uliginosum fruits harvested in different world areas, some important differences were observed.

Changes in polyphenol and sugar concentrations in wild type and genetically modified Nicotiana langsdorffii Weinmann in response to water and heat stress.

In this study wild type Nicotiana langsdorffii plants were genetically transformed by the insertion of the rat gene (gr) encoding the glucocorticoid receptor or the rolC gene and exposed to water and heat stress. Water stress was induced for 15 days by adding 20% PEG 6000 in the growth medium, whereas the heat treatment was performed at 50 °C for 2 h, after that a re-growing capability study was carried out. The plant response to stress was investigated by determining electrolyte leakage, dry weight biomass production and water content. These data were evaluated in relation to antiradical activity and concentrations of total polyphenols, selected phenolic compounds and some soluble sugars, as biochemical indicators of metabolic changes due to gene insertion and/or stress treatments. As regards the water stress, the measured physiological parameters evidenced an increasing stress level in the order rolC < gr < WT plants (e.g. about 100% and 50% electrolyte leakage increase in WT and gr samples, respectively) and complied with the biochemical pattern, which consisted in a general decrease of antiradical activity and phenolics, together with an increase in sugars. As regard heat stress, electrolyte leakage data were only in partial agreement with the re-growing capability study. In fact, according to this latter evaluation, gr was the genotype less affected by the heat shock. In this regard, sugars and especially phenolic compounds are informative of the long-term effects due to heat shock treatment.

Chromium accumulation and changes in plant growth, selected phenolics and sugars of wild type and genetically modified Nicotiana langsdorffii.

Chromium accumulation, dry weight (DW) biomass yield, water content and concentrations of some selected phenolic compounds and carbohydrates were determined in root and shoot of Nicotiana langsdorffii, either wild type (WT) or genetically modified by the insertion of GR and rolC genes, in response to the presence of Cr(VI) in the growth medium. A biomass decrease was observed for WT plants, but not for GR and rolC transformations, in response to Cr(VI) in the growth medium, highlighting a stress situation only in WT line. Shoot chromium concentrations were in all cases about 300 mg kg(-1) DW. In root higher concentrations were found in rolC than in GR and WT (3843, 2600 and 2751 mg kg(-1) DW, respectively). Based on the DW biomass, GR and WT accumulated higher chromium quantities than rolC, both in root (330 and 424 versus 85 µg Cr per plant) and shoot (282 and 275 versus 121 µg Cr per plant). Therefore, GR should be preferred to WT as a promising candidate for chromium phytoremediation. Metabolic shifts of sugars and phenolics were generally observed in response to either gene insertions or exposure to Cr(VI), being the latter more related to the resistance to Cr(VI) than the former.