A novel black poplar propolis extract with promising health-promoting properties: focus on its chemical composition, antioxidant, anti-inflammatory, and anti-genotoxic activities.

Propolis is a resinous mixture produced by honeybees which has been used since ancient times for its useful properties. However, its chemical composition and bioactivity may vary, depending on the geographical area of origin and the type of tree bees use for collecting pollen. In this context, this research aimed to investigate the total phenolic content (using the Folin-Ciocalteu assay) and the total antioxidant capacity (using the FRAP, DPPH, and ABTS assays) of three black poplar (Populus nigra L.) propolis (BPP) solutions (S1, S2, and S3), as well as the chemical composition (HPLC-ESI-MSn) and biological activities (effect on cell viability, genotoxic/antigenotoxic properties, and anti-inflammatory activity, and effect on ROS production) of the one which showed the highest antioxidant activity (S1). The hydroalcoholic BPP solution S1 was a prototype of an innovative, research-type product by an Italian nutraceutical manufacturer. In contrast, hydroalcoholic BPP solutions S2 and S3 were conventional products purchased from local pharmacy stores. For the three extracts, 50 phenolic compounds, encompassing phenolic acids and flavonoids, were identified. In summary, the results showed an interesting chemical profile and the remarkable antioxidant, antigenotoxic, anti-inflammatory and ROS-modulating activities of the innovative BPP extract S1, paving the way for future research. In vivo investigations will be a possible line to take, which may help corroborate the hypothesis of the potential health benefits of this product, and even stimulate further ameliorations of the new prototype.

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.

Estimating the hydrophobicity extent of molecular fragments using reversed-phase liquid chromatography.

A fast HPLC method was developed to study the hydrophobicity extent of pharmaceutically relevant molecular fragments. By this strategy, the reduced amount of sample available for physico-chemical evaluations in early-phase drug discovery programs does not represent a limiting factor. The sixteen acid fragments investigated were previously synthesized also determining potentiometrically their experimental log D values. For four fragments it was not possible to determine such property since their values were outside of the instrumental working range (2 < pKa  < 12). An RP-HPLC method was therefore optimized. For each scrutinized method, some derived chromatographic indices were calculated, and Pearson's correlation coefficient (r) allowed to select the so-called "φ0 index" as the best correlating with the log D. The w s p H ${}_w/pH$ was fixed at 3.5 and a modification of some variables [organic modifier (methanol vs. ACN), stationary phase (octyl vs. octadecyl), presence/absence of the additives n-octanol, n-butylamine, and n-octylamine], allowed to select the best correlation conditions, producing a r = 0.94 (p < 0.001). Importantly, the φ0 index enabled the estimation of log D values for four fragments which were unattainable by potentiometric titration. Moreover, a series of molecular descriptors were calculated to identify the chemical characteristics of the fragments explaining the obtained φ0 . The number of hydrogen bond donors and the index of cohesive interaction correlated with the experimental data.

Microsampling and enantioselective liquid chromatography coupled to mass spectrometry for chiral bioanalysis of novel psychoactive substances.

In this paper, the development of efficient enantioselective HPLC methods for the analysis of five benzofuran-substituted phenethylamines, two substituted tryptamines, and three substituted cathinones is described. For the first time, reversed-phase (eluents made up with acidic water-methanol solutions) and polar-ionic (eluent made up with an acetonitrile-methanol solution incorporating both an acidic and a basic additive) conditions fully compatible with mass spectrometry (MS) detectors were applied with a chiral stationary phase (CSP) incorporating the (+)-(18-crown-6)-tetracarboxylic acid chiral selector. Enantioresolution was achieved for nine compounds with α and RS factors up to 1.32 and 5.12, respectively. Circular dichroism (CD) detection, CD spectroscopy in stopped-flow mode and quantum mechanical (QM) calculations were successfully employed to investigate the absolute stereochemistry of mephedrone, methylone and butylone and allowed to establish a (R)<(S) enantiomeric elution order for these compounds on the chosen CSP. Whole blood miniaturized samples collected by means of volumetric absorptive microsampling (VAMS) technology and fortified with the target analytes were extracted following an optimized protocol and effectively analysed by means of an ultra-high performance liquid chromatography-MS system. By this way a proof-of-concept procedure was applied, demonstrating the suitability of the method for quali-quantitative enantioselective assessment of the selected psychoactive substances in advanced biological microsamples. VAMS microsamplers including a polypropylene handle topped with a small tip of a polymeric porous material were used and allowed to volumetrically collect small aliquots of whole blood (10 µL) independently from its density. Highly appreciable volumetric accuracy (bias, in the -8.7-8.1% range) and precision (% CV, in the 2.8-5.9% range) turned out.

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.

Elucidation of retention mechanism of dipeptides on a ristocetin A-based chiral stationary phase using a combination of chromatographic and molecular simulation techniques.

Two chiral stationary phases virtually reproducing the Nautilus-R column were modeled in silico to study the enantiorecognition mechanism of some selected dipeptides, taking into consideration the two different anchoring alternatives to the silica layer involving the two ristocetin A amino groups. A mobile phase composed of water-methanol (40:60, v/v) was included in the system. The analyses of the trajectories supported the experimental L(LL)

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.

Efficient enantioresolution of aromatic α-hydroxy acids with Cinchona alkaloid-based zwitterionic stationary phases and volatile polar-ionic eluents.

Single enantiomers of mandelic acid (1), 3-phenyllactic acid (2), and 3-(4-hydroxyphenyl)lactic acid (3) are the subject of many fields of investigation, spanning from the pharmaceutical synthesis to that of biocompatible and biodegradable polymers, while passing from the interest towards their antimicrobial activity to their role as biomarkers of particular pathological conditions or occupational exposures to specific xenobiotics. All above mentioned issues justify the need for accurate analytical methods enabling the correct determination of the individual enantiomers. So far, all the developed liquid chromatography (LC) methods were not or hardly compatible with mass spectrometry (MS) detection. In this paper, a commercially available Cinchona-alkaloid derivative zwitterionic chiral stationary phase [that is, the CHIRALPAK® ZWIX(-)] was successfully used to optimize the enantioresolution of compounds 1-3 under polar-ionic (PI) conditions with a mobile phase consisting of an acetonitrile/methanol 95/5 (v/v) mixture with 80 mM formic acid. With the optimized conditions, enantioseparation and enantioresolution values up to 1.46 and 4.41, respectively, were obtained. In order to assess the applicability of the optimized enantioselective chromatography conditions in real-life scenarios and on MS-based systems, a proof-of-concept application was efficiently carried out by analysing dry urine spot samples spiked with 1 by means of a LC-MS system. The (S)<(R) enantiomer elution order (EEO) was established for compounds 1 and 2 by analysing a pure enantiomeric standard of known configuration. This was not possible for 3 because not commercially available. For this compound, the same EEO was identified applying a procedure based on ab initio time-dependent density-functional theory simulations coupled to electronic circular dichroism analyses. Moreover, a molecular dynamics simulation unveiled the role of the phenolic OH in compound 3 in the retention mechanism.

Original enantioseparation of illicit fentanyls with cellulose-based chiral stationary phases under polar-ionic conditions.

Fentanyl analogues used in therapy and a range of highly potent non-pharmaceutical fentanyl derivatives are subject to international control, as the latter are increasingly being synthesized illicitly and sold as 'synthetic heroin', or mixed with heroin. A significant number of hospitalizations and deaths have been reported in the EU and USA following the use of illicitly synthesized fentanyl derivatives. It has been unequivocally demonstrated that the enantiomers of fentanyl derivatives exhibit different pharmaco-toxicological profiles, which makes crucial to avail of suitable analytical methods enabling investigations at a "stereochemical level". Chromatographic methods useful to discriminate the enantioseparation of fentanyls and their derivatives are still missing in the literature. This is the first study in which the enantioseparation of four fentanyl derivatives, that is, (±)-trans-3-methyl norfentanyl, (±)-cis-3-methyl norfentanyl, ß-hydroxyfentanyl, and ß-hydroxythiofentanyl, has been obtained under polar-ionic conditions. Indeed, the use of ACN-based mobile phases with minor amounts of either 2-propanol or ethanol (plus diethylamine and formic acid as ionic additives) allowed obtaining enantioseparation and enantioresolution factors up to 1.83 and 7.02, respectively. For the study, the two chiral stationary phases cellulose tris(3-chloro-4-methylphenylcarbamate) and cellulose tris(4-chloro-3-methylphenylcarbamate) were used, displaying a remarkably different performance towards the enantioseparation of (±)-cis-3-methyl norfentanyl. Chiral LC analyses with a high-resolution mass spectrometry detector were also carried out in order to confirm the obtained data and demonstrate the suitability and compatibility of the optimized mobile phases with mass spectrometric systems.

Enantioselective HPLC Analysis to Assist the Chemical Exploration of Chiral Imidazolines.

In the present work, we illustrate the ability of high-performance liquid chromatography (HPLC) analysis to assist the synthesis of chiral imidazolines within our medicinal chemistry programs. In particular, a Chiralpak® IB® column containing cellulose tris(3,5-dimethylphenylcarbamate) immobilized onto a 5 µm silica gel was used for the enantioselective HPLC analysis of chiral imidazolines synthesized in the frame of hit-to-lead explorations and designed for exploring the effect of diverse amide substitutions. Very profitably, reversed-phase (RP) conditions succeeded in resolving the enantiomers in nine out of the 10 investigated enantiomeric pairs, with α values always higher than 1.10 and RS values up to 2.31. All compounds were analysed with 50% (v) water while varying the content of the two organic modifiers acetonitrile and methanol. All the employed eluent systems were buffered with 40 mM ammonium acetate while the apparent pH was fixed at 7.5. Based on the experimental results, the prominent role of π-π stacking interactions between the substituted electron-rich phenyl groups outside of the polymeric selector and the complementary aromatic region in defining analyte retention and stereodiscrimination was identified. The importance of compound polarity in explaining the retention behaviour with the employed RP system was readily evident when a quantitative structure-property relationship study was performed on the retention factor values (k) of the 10 compounds, as computed with a 30% (v) methanol containing mobile phase. Indeed, good Pearson correlation coefficients of retention factors (r - log k1st = -0.93; r - log k2nd = -0.94) were obtained with a water solubility descriptor (Ali-logS). Interestingly, a n-hexane/chloroform/ethanol (88:10:2, v/v/v)-based non-standard mobile phase allowed the almost base-line enantioseparation (α = 1.06; RS = 1.26) of the unique compound undiscriminated under RP conditions.