Development of an easy-to-set-up multiple heart-cutting achiral-chiral LC-LC method for the analysis of branched-chain amino acids in commercial tablets.

In this paper, the development and application of a multiple heart-cutting achiral-chiral LC-LC method (mLC-LC) for the analysis of dansylated (Dns) branched-chain amino acids in commercial tablets are described. In the first dimension, a Waters Xbridge RP C18 achiral column was used under gradient conditions with buffered aqueous solution and acetonitrile. The elution order Dns-valine (Dns-Val) < Dns-isoleucine (Dns-Ile) < Dns-leucine (Dns-Leu) turned out with full resolution between adjacent peaks: 7.25 and 1.50 for the Val/Ile and the Ile/Leu pairs, respectively. A "research" validation study was performed, revealing high accuracy (Recovery%) and precision (RSD%) using two external set solutions, respectively, in the range 93.7%-104.1% and 0.4%-3.2%. The C18 column was connected via a two-position six-port switching valve to the quinidine-based Chiralpak quinidine-anion-exchange chiral column. A water/acetonitrile, 30/70 (v/v) with 50 mM ammonium acetate (apparent pH of 5.5) eluent allowed getting the three enantiomers' pairs resolved: RS equal to 4.3 for Dns-Val and Dns-Ile, and 1.7 for Dns-Leu. The application of the mLC-LC method confirmed that the content of Val, Ile, and Leu in the tablets was compliant with that labeled by the producer. Only l-enantiomers were found in the food supplement, as confirmed by LC-MS/MS analysis.

Environmentally Sustainable Achiral and Chiral Chromatographic Analysis of Amino Acids in Food Supplements.

Two LC methods were developed for the achiral and chiral reversed-phase (RP) analysis of an amino acid (AA) pool in a food supplement, in compliance with the main paradigms of Green Chromatography. A direct achiral ion-pairing RP-HPLC method was optimized under gradient conditions with a water-ethanol (EtOH) eluent containing heptafluorobutyric acid (0.1%, v/v), to quantify the eight essential AAs (Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val) contained in the food supplement. Thus, the usually employed acetonitrile was profitably substituted with the less toxic and more benign EtOH. The method was validated for Leu and Phe. The chiral LC method performed with a teicoplanin chiral stationary phase was developed with a water-EtOH (60:40, v/v) eluent with 0.1%, v/v acetic acid. The enantioselective analysis was carried out without any prior derivatization step. Both developed methods performed highly for all eight AAs and revealed that: (i) the content of six out of eight AAs was consistent with the manufacturer declaration; (ii) only L-AAs were present. Furthermore, it was demonstrated that a two-dimensional achiral-chiral configuration is possible in practice, making it even more environmentally sustainable. A molecular modelling investigation revealed interesting insights into the enantiorecognition mechanism of Lys.

In-depth characterization of phenolic profiling of Moraiolo extra-virgin olive oil extract and initial investigation of the inhibitory effect on Indoleamine-2,3-Dioxygenase (IDO1) enzyme.

In this research, the phenolic extract from Moraiolo extra-virgin olive oil (EVOO) was thoroughly characterized. A reversed-phase HPLC method with a photodiode detector allowed to measure a total phenol content higher than 500 mg/kg EVOO, with elevated amounts of oleocanthal, oleacein, and oleouropein aglycone (131.2, 213.5, and 158.4 mg/kg EVOO, respectively). Appreciable amounts of (+)-pinoresinol and (+)- 1-acetoxy-pinoresinol, 3.2 and 12.5 mg/kg EVOO respectively, were measured. High-resolution mass spectrometry with Orbitrap mass analyzer technology was used to confirm the identity of the analytes. Afterwards, the extract was tested, for the first time, for its activity on Indoleamine-2,3-Dioxygenase (IDO1). This enzyme appears as a promising target for the modulation of the neuroinflammatory-oxidative processes relying on the pathogenesis of several neurodegenerative diseases. The extract showed an inhibitory effect on the catalytic activity of both human and murine IDO1, with a good safety at the concentrations of 15 and 30 µg/mL.

Separation and determination of cysteine enantiomers in plasma after derivatization with 4-fluoro-7-nitrobenzofurazan.

The importance of D-amino acids in mammals associated with enantio-dependent biological functions has been increasingly highlighted. In addition to naturally occurring, D-amino acid supplementation could have a positive biological impact, including cytoprotective implications. In this context, supplementation with D-cysteine has revealed beneficial effects. Quantification of cysteine enantiomers in rodent plasma has been achieved by using 4-fluoro-7-nitrobenzofurazan derivatization of the target analytes. Cystine, the main form of cysteine in the plasma, was initially reduced to cysteine using DL-dithiothreitol. Baseline enantioseparation was then achieved in less than 3 min using a (R,R)-Whelk-O 1 stationary phase and isocratic elution using CH3OH-H2O 90:10 (v/v) with 15 mM ammonium formate (apparent pH 6.0) at 0.5 mL/min. The derivatives were then detected using negative ESI-MS in SRM mode. An external calibration was employed for D-cysteine, while L-cysteine quantification, as an endogenous analyte, was addressed using a background subtraction strategy. The method was validated. Response functions were obtained from 0 to 300 µM and from 0 to 125 µM for D-cysteine and L-cysteine, respectively. The trueness ranged from 96% to 105% for both enantiomers with repeatability and intermediate precision lower than 8% and 15% for the D-form and the endogenous L-form, respectively. The method was successfully applied for determining D- and L-cysteine in mouse plasma after D-cysteine administration.

Optimized one-pot derivatization and enantioseparation of cysteine: Application to the study of a dietary supplement.

Cysteine is a sulfur-containing amino acid which plays an outstanding role in many biological pathways in mammals. The analysis and quantification of native cysteine remains a critical issue due to its highly reactive thiol group evolving to the disulfide cystine derivative through oxidation reaction. Aimed at improving the derivative stability, cysteine was labelled with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), which reacts with both amino and thiol groups. The derivatization was optimized and the chemical identity of the reaction product was assessed via high-resolution mass spectrometry. The NBD-cysteine derivative resulted stable for 10 days. This derivative was enantioresolved (α and RS equal to 1.25 and 2.70, respectively) thanks to a (R,R)-Whelk-O1 phase with the following chromatographic setting: eluent, MeOH/water-90/10 (v/v) with 15 mM ammonium formate (pwsH 6.0); column temperature, 35 °C; flow rate, 1.0 mL/min. The developed method was validated following the ICH guidelines and applied for the quality control of a L-cysteine containing dietary supplement.

Development and validation of a HPLC method for the direct separation of carnosine enantiomers and analogues in dietary supplements.

The chiral purity of some molecules such as nutraceuticals is fundamental to ensure their beneficial activities and it must be checked during quality control analysis. Carnosine is a natural histidine dipeptide used as ingredient for food supplements, but only his L-enantiomer is absorbed and active. Despite of this feature, a method for the separation of carnosine enantiomers without derivatization has only recently been published. Herein, we validated a method based on a Chirobiotic T column and an UV detector for the direct quantification of carnosine enantiomers, following ICH guideline. Moreover, we demonstrated that elution with water containing 0.1% formic acid and 20-40% ensures stereo-, chemo- and regio-selectivity for the separation and the identification of carnosine enantiomers and natural analogues. Moreover, the method allows a direct hyphenation with electrospray mass spectrometry to increase detection selectivity and sensitivity. As far as we know, this is the first method allowing the simultaneous identification and quantification of natural analogues of carnosine, which can be important for application such as the identification of enantiomeric impurities or adulteration that can occur during the storage or the preparation of foods or food supplements containing histidine dipeptides.

Onion (Allium cepa L.) Skin: A Rich Resource of Biomolecules for the Sustainable Production of Colored Biofunctional Textiles.

The aqueous extract of dry onion skin waste from the 'Dorata di Parma' cultivar was tested as a new source of biomolecules for the production of colored and biofunctional wool yarns, through environmentally friendly dyeing procedures. Specific attention was paid to the antioxidant and UV protection properties of the resulting textiles. On the basis of spectrophotometric and mass spectrometry analyses, the obtained deep red-brown color was assigned to quercetin and its glycoside derivatives. The Folin⁻Ciocalteu method revealed good phenol uptakes on the wool fiber (higher than 27% for the textile after the first dyeing cycle), with respect to the original total content estimated in the water extract (78.50 ± 2.49 mg equivalent gallic acid/g onion skin). The manufactured materials showed remarkable antioxidant activity and ability to protect human skin against lipid peroxidation following UV radiation: 7.65 ± 1.43 (FRAP assay) and 13.60 (ORAC assay) mg equivalent trolox/g textile; lipid peroxidation inhibition up to 89.37%. This photoprotective and antioxidant activity were therefore ascribed to the polyphenol pool contained in the outer dried gold skins of onion. It is worth noting that citofluorimetric analysis demonstrated that the aqueous extract does not have a significative influence on cell viability, neither is capable of inducing a proapoptotic effect.

Hydrophobic Amino Acid Content in Onions as Potential Fingerprints of Geographical Origin: The Case of Rossa da Inverno sel. Rojo Duro.

In this study, we were interested in comparing the amino acid profile in a specific variety of onion, Rossa da inverno sel. Rojo Duro, produced in two different Italian sites: the Cannara (Umbria region) and Imola (Emilia Romagna region) sites. Onions were cultivated in a comparable manner, mostly in terms of the mineral fertilization, seeding, and harvesting stages, as well as good weed control. Furthermore, in both regions, the plants were irrigated by the water sprinkler method and subjected to similar temperature and weather conditions. A further group of Cannara onions that were grown by micro-irrigation was also evaluated. After the extraction of the free amino acid mixture, an ion-pairing reversed-phase (IP-RP) HPLC method allowed for the separation and the evaporative light scattering detection of almost all the standard proteinogenic amino acids. However, only the peaks corresponding to leucine (Leu), phenylalanine (Phe), and tryptophan (Trp), were present in all the investigated samples and they were unaffected from the matrix interfering peaks. The use of the beeswarm/box plots revealed that the content of Leu and Phe were markedly influenced by the geographical origin of the onions (with *** p.

N-Decyl-S-trityl-(R)-cysteine, a new chiral selector for "green" ligand-exchange chromatography applications.

In search for new enantioselectivity profiles, the N-decyl-S-trityl-(R)-cysteine [C10-(R)-STC] was synthesized through a one-step procedure and then hydrophobically adsorbed onto an octadecylsilica surface to generate a stable chiral stationary phase for ligand-exchange chromatography (CLEC-CSP) applications. The CLEC analysis was carried out on underivatized amino acids, by using a Cu(II) sulphate (1.0mM) containing aqueous eluent system. Most of the analysed compounds (34 out of 45) were enantiodiscriminated by the C10-(R)-STC-based CSP, with resolution factor (RS) values up to 8.86. Conformationally rigid and hydrophobic ligands often experienced the largest enantioselectivity effects. A high loadability emerged from the analysis of rac-NorVal (selected as prototype test compound): up to 20mg/mL were efficiently enantioseparated with the CLEC-CSP. Two in-line hand-made cartridges filled with a strong cation-exchange resin allowed the effective catching of Cu(II) ions after the semi-preparative enantioseparation. The quantitative recovery of the rac-NorVal enantiomers was made possible by flowing through the cartridge a 5% (v) ammonia solution. The CLEC phase proved successful in the enantioselective analysis of a commercially available (S)-Leu containing tablet. Furthermore, in order to understand the molecular basis for a successful use of the C10-(R)-STC-based CLEC system, a descriptive structure-separation relationship study was performed. As a result, all compounds with a MEAN-QPlogS (a hydrophilicity descriptor) value lower than 0.373 can be most likely enantioseparated with the CLEC system under investigation. In the work, the numerous aspects complying with the principles of green chromatography are highlighted and discussed.

Quinine-Based Zwitterionic Chiral Stationary Phase as a Complementary Tool for Peptide Analysis: Mobile Phase Effects on Enantio- and Stereoselectivity of Underivatized Oligopeptides.

Peptide stereoisomer analysis is of importance for quality control of therapeutic peptides, the analysis of stereochemical integrity of bioactive peptides in food, and the elucidation of the stereochemistry of peptides from a natural chiral pool which often contains one or more D-amino acid residues. In this work, a series of model peptide stereoisomers (enantiomers and diastereomers) were analyzed on a zwitterionic ion-exchanger chiral stationary phase (Chiralpak ZWIX(+) 5 µm), in order to investigate the retention and separation performance for such compounds on this chiral stationary phase and elucidate its utility for this purpose. The goal of the study focused on 1) investigations of the effects of the sample matrix used to dissolve the peptide samples; 2) optimization of the mobile phase (enabling deriving information on factors of relevance for retention and separation); and 3) derivation of structure-selectivity relationships. It turned out that small di- and tripeptides can be well resolved under optimized conditions, typically with resolutions larger than 1.5. The optimized mobile phase often consisted of methanol-tetrahydrofuran-water (49:49:2; v/v/v) with 25 mM formic acid and 12.5 mM diethylamine. This work proposes some guidance on which mobile phases can be most efficiently used for peptide stereoisomer separations on Chiralpak ZWIX. Chirality 28:5-16, 2016. © 2015 Wiley Periodicals, Inc.

Antioxidant activity of phenolic extracts from different cultivars of Italian onion (Allium cepa) and relative human immune cell proliferative induction.

CONTEXT: The total antioxidant activity (TAC) may vary considerably between onion cultivars. Immunological effects of onion phenolic compounds are still underestimated. OBJECTIVE: The objective of this study is to determine the total phenol content (TPC) and the relative TAC of three Allium cepa L. (Liliaceae) onion cultivars cultivated in Cannara (Italy): Rossa di Toscana, Borettana di Rovato, and Dorata di Parma, and to evaluate the phenol extracts ability to induce human immune cell proliferation. MATERIALS AND METHODS: TPC was determined by the Folin-Ciocalteu method, TAC with FRAP, TEAC/ABTS, and DPPH methods. Peripheral blood mononuclear cells from healthy human donors were incubated for 24 h at 37 °C with 1 ng/mL of phenolic extract in PBS, immunostained, and then analyzed by 4-color flow cytometry for the phenotypic characterization of T helper cells (CD4+ cells), cytotoxic T lymphocytes (CD8+ cells), T regulatory cells (CD25high CD4+ cells), and natural killer cells/monocytes (CD16+ cells). RESULTS: Rossa di Toscana displayed the highest TPC (6.61 ± 0.87 mg GA equivalents/g onion bulb DW) and the highest TAC with the experienced methods: FRAP, 9.19 ± 2.54 µmol Trolox equivalents/g onion bulb DW; TEAC/ABTS, 21.31 ± 0.41 µmol Trolox equivalents/g onion bulb DW; DPPH, 22.90 ± 0.01 µmol Trolox equivalents/g onion bulb DW. Incubation with Rossa di Toscana extract determined an increase in the frequency of the antitumor/anti-infection NK CD16+ immune cells (23.0 ± 0.4%). DISCUSSION AND CONCLUSIONS: Content of health-promoting phenols and the deriving antioxidant and immunostimulating activity vary considerably among the investigated cultivars. Rossa di Toscana can be considered as a potential functional food.

Pyrazole[3,4-e][1,4]thiazepin-7-one derivatives as a novel class of Farnesoid X Receptor (FXR) agonists.

A virtual screening procedure was applied to the discovery of structurally diverse non-steroidal Farnesoid X Receptor (FXR) agonists. From 117 compounds selected by virtual screening, a total of 47 compounds were found to be FXR agonists, with 34 of them showing activity below a concentration of 20 µM. 1H-Pyrazole[3,4-e][1,4]thiazepin-7-one-based hit compound 7 was chosen for hit-to-lead optimization. A large number of 1H-pyrazole[3,4-e][1,4]thiazepin-7-one derivatives was designed, synthesized, and evaluated by a cell-based luciferase transactivation assay for their agonistic activity against FXR. Most of them exhibited low micromolar range of potency and very high efficacy.