Evaluation of different QuEChERS-based methods for the extraction of 48 wastewater-derived organic contaminants from soil and lettuce root using high-resolution LC-QTOF with MRMHR and SWATH acquisition modes.

The reuse of treated wastewater in agriculture is an important route of introducing a large number of organic contaminants into the agroecosystem. In this study, a modified QuEChERS-based approach was developed for rapid, simple, and simultaneous extraction of 48 organic wastewater-derived contaminants from soil and lettuce root. Twenty-two different (modification) scenarios of the known (or original) QuEChERS method have been tested, in order to obtain best and well-compromised recoveries for all target compounds for soil and roots. Finally, a common method was chosen for both matrices consisting of a single extraction step using EDTA-Mcllvaine buffer and the unbuffered Original QuEChERS salts. Method performance was accomplished by liquid chromatography coupled with high-resolution mass spectrometry on a QToF-MS system using two different acquisition modes, the ultra-fast high-resolution multiple reaction monitoring (MRMHR) mode and the innovative Sequential Window Acquisition of All Theoretical Fragment-Ion (SWATH) mode. Performance characterization was evaluated in terms of recovery, linearity, intra-day precision, method detection limits (MDLs), method quantification limits (MQLs), and matrix effect (ME). Recoveries in MRMHR mode ranged from 63 to 111% and 54 to 104% for lettuce root and soil, respectively, for most of compounds in MRMHR mode and from 56 to 121% and 54 to 104% for lettuce root and soil, respectively, for most of compounds in SWATH. Whereas, MQLs ranged from 0.03 to 0.92 ng g-1 in MRMHR and from 0.03 to 82 ng g-1 in SWATH for lettuce root, and from 0.02 to 0.44 ng g-1 in MRMHR and 0.02 to 0.14 ng g-1 in SWATH for soil. The method was then applied to follow the target compounds in soil and lettuce root, where the system lettuce-soil was irrigated with treated wastewater under real greenhouse conditions. Five and 17 compounds were detected in lettuce root and soil, respectively.

High-speed countercurrent chromatography with offline detection by electrospray mass spectrometry and nuclear magnetic resonance detection as a tool to resolve complex mixtures: A practical approach using Coffea arabica leaf extract.

INTRODUCTION: Many secondary metabolites isolated from plants have been described in the literature owing to their important biological properties and possible pharmacological applications. However, the identification of compounds present in complex plant extracts has remained a great scientific challenge, is often laborious, and requires a long research time with high financial cost. OBJECTIVES: The aim of this study was to develop a method that allows the identification of secondary metabolites in plant extracts with a high degree of confidence in a short period of time. MATERIAL AND METHODS: In this study, an ethanolic extract of Coffea arabica leaves was used to validate the proposed method. Countercurrent chromatography was chosen as the initial step for extraction fractionation using gradient elution. Resulting fractions presented a variation of compounds concentrations, allowing for statistical total correlation spectroscopy (STOCSY) calculations between liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-HRMS/MS) and NMR across fractions. RESULTS: The proposed method allowed the identification of 57 compounds. Of the annotated compounds, 20 were previously described in the literature for the species and 37 were reported for the first time. Among the inedited compounds, we identified flavonoids, alkaloids, phenolic acids, coumarins, and terpenes. CONCLUSION: The proposed method presents itself as a valid alternative for the study of complex extracts in an effective, fast, and reliable way that can be reproduced in the study of other extracts.

Evaluation of antiproliferative activity, apoptotic induction and LC-HRMS/MS analyses of the VLC fractions of L. numidicum.

In this study, six vacuum liquid chromatography (VLC) fractions (F1-F6) of the n-BuOH extract of L. numidicum Murb. (BELN) were examined for their anticancer capacity. The composition of secondary metabolites was analyzed by LC-HRMS/MS. The antiproliferative effect against PC3 and MDA-MB-231 lines was evaluated by MTT assay. Apoptosis of PC3 cells was detected by annexin V-FITC/PI staining using a flow cytometer. The results showed that only fractions 1 and 6 inhibited PC3 and MDA-MB 231 cell proliferation in a dose-dependent manner and induced dose-dependent apoptosis of PC3 cells, evidenced by the accumulation of early and late apoptotic cells, and by the decrease in viable cells. LC-HRMS/MS profiling of fractions 1 and 6 revealed the presence of known compounds that may be responsible for the observed anticancer activity. F1 and F6 may be an excellent source of active phytochemicals for cancer treatment.

LC-HRMS/MS phytochemical profiling of Symphytum officinale L. and Anchusa ochroleuca M. Bieb. (Boraginaceae): Unveiling their multi-biological potential via an integrated approach.

This study was aimed at providing a comprehensive phytochemical characterization and multi-biological assessment of Symphytum officinale L., a medicinal plant with a noteworthy traditional use, and Anchusa ochroleuca M. Bieb., a Boraginaceae species from the Romanian flora. The dichloromethane, methanol and 65 % ethanol extracts obtained from the roots and aerial parts of both plants revealed the presence of numerous phenolic acids, oxygenated fatty acids, pyrrolizidine alkaloids (PAs) and flavonoids, as assessed by LC-HRMS/MS analysis. Consistent with their higher total phenolic content, the polar aerial part extracts of S. officinale and root extracts of A. ochroleuca showed the most significant antioxidant activities, as evaluated by DPPH (173.22-216.98 mg TE/g) and ABTS (219.41-311.97 mg TE/g) radical scavenging, CUPRAC (387.18-626.40 mg TE/g), FRAP (199.36-299.86 mg TE/g) and total antioxidant capacity (2.28-2.68 mmol TE/g). Furthermore, both plants exhibited good tyrosinase (19.11-43.89 mg KAE/g) and α-glucosidase (2.45-12.54 mmol ACAE/g) inhibitory effects. The orthogonal projections to latent structures discriminant analysis (OPLS-DA) allowed the objective differentiation between the roots and aerial parts of the two investigated species based on their phytochemical and biological profiles. The partial least square (PLS) analysis showed that several individual phenolic acids, such as danshensu, rabdosiin and rosmarinic acid, significantly contributed to the antioxidant potential of both Boraginaceae species, whilst the relative levels of sucrose were positively correlated with the anti-enzymatic properties. Overall, S. officinale and A. ochroleuca could be regarded as rich sources of bioactive phytochemicals that could further lead to developing novel phyto-pharmaceutical commodities.

Metabolomic Profiling and Toxicokinetics Modeling to Assess the Effects of the Pharmaceutical Diclofenac in the Aquatic Invertebrate Hyalella azteca.

The exposure of ecologically critical invertebrate species to biologically active pharmaceuticals poses a serious risk to the aquatic ecosystem. Yet, the fate and toxic effects of pharmaceuticals on these nontarget aquatic invertebrates and the underlying mechanisms are poorly studied. Herein, we investigated the toxicokinetic (TK) processes (i.e., uptake, biotransformation, and elimination) of the pharmaceutical diclofenac and its biotransformation in the freshwater invertebrate Hyalella azteca. We further employed mass spectrometry-based metabolomics to assess the toxic effects of diclofenac on the metabolic functions of H. azteca exposed to environmentally relevant concentrations (10 and 100 µg/L). The TK results showed a quick uptake of diclofenac by H. azteca (maximum internal concentration of 1.9 µmol/kg) and rapid formation of the conjugate diclofenac taurine (maximum internal concentration of 80.6 µmol/kg), indicating over 40 times higher accumulation of diclofenac taurine than that of diclofenac in H. azteca. Depuration kinetics demonstrated that the elimination of diclofenac taurine was 64 times slower than diclofenac in H. azteca. Metabolomics results suggested that diclofenac inhibited prostaglandin synthesis and affected the carnitine shuttle pathway at environmentally relevant concentrations. These findings shed light on the significance of the TK process of diclofenac, especially the formation of diclofenac taurine, as well as the sublethal effects of diclofenac on the bulk metabolome of H. azteca. Combining the TK processes and metabolomics provides complementary insights and thus a better mechanistic understanding of the effects of diclofenac in aquatic invertebrates.

Chestnut shells (Italian cultivar "Marrone di Roccadaspide" PGI): Antioxidant activity and chemical investigation with in depth LC-HRMS/MSn rationalization of tannins.

In Italy a particularly valuable chestnut is "Marrone di Roccadaspide", a protected geographical indication (PGI) product, deriving from a Castanea sativa cultivar, typical of Salerno province in Campania region. As chestnut industrial processes yield a large amount of shell by-products, in this study the possibility to retrain this waste food as potential source of bioactives was investigated. The ability of "Marrone di Roccadaspide" shell MeOH extract to modulate the pro-inflammatory transcriptional factor NF-κB after LPS stimulation, along with the antioxidant activity by a cell-based in vitro test, were evaluated. To correlate the NF-κB inhibition (67.67% at 5 µg/mL) and the strong antioxidant activity to the chemical composition, an analytical approach based on LC-ESI/LTQOrbitrap/MS/MSn along with NMR characterization of isolated compounds was developed. The identification of hydrolysable and condensed tannins, along with flavonoids, phenol glucosides, ellagic acid derivatives, and triterpenoids was accomplished. The most representative compounds were quantitatively analyzed by LC-ESI/QTrap/MS/MS, showing bartogenic acid as the compound occurring in the highest amount (103.08 mg/100 g shells). With the aim to explore the possibility to employ chestnut shells as suitable source of bioactives for the preparation of functional ingredients, the chemical composition and the antioxidant activity of "eco-friendly" extracts (EtOH and EtOH:H2O 7:3) was finally evaluated, showing a high superimposability of the EtOH:H2O (7:3) extract to the MeOH extract.