Influence of Post-Harvest 1-Methylcyclopropene (1-MCP) Treatment and Refrigeration on Chemical Composition, Phenolic Profile and Antioxidant Modifications during Storage of Abate Fétel Pears.

'Abate Fétel', a winter cultivar, is the most important pear cultivar in Italy; its fruits are appreciated by consumers for their aroma, texture and balanced sweet and sour taste. Maintaining high-quality characteristics to prolong the shelf-life of fruit and preserve the sensory and nutritional quality is a priority for the food industry. The aim of our study was to test the effectiveness of 1-methylcyclopropene (1-MCP) and cold storage in prolonging the shelf-life of these fruits, which were harvested at maturity at two different times. This work focused on the effects of different storage treatments and two ripening times on (i) the chemical composition of Abate Fétel pulp fruits to preserve their sweet taste and aroma and (ii) the phenolic profile composition and antioxidant activity of the peel, which is naturally rich in phytochemicals and important for the fruit's shelf-life and in the functional food industry for its high nutritional value. Abate Fétel fruits were harvested at the optimal commercial maturity stage, first on 15 September, having been treated with 1-MCP and stored for 2 months at cold temperatures; the other fruits were harvested at the end of September and stored in a cold cell for 2 months. The fruit pulp was tested for glucose and fructose, pH, acidity and organic acids (malic, citric, fumaric and shikimic), phenolic content and phenolic compounds (chlorogenic and caffeic acids, rutin, hyperoside, kaempferol-3-rutinoside and isoquercitrin), and the antioxidant activities in the fruit peels were measured. Treating the fruits with 1-MCP better preserved the phytochemical compounds compared to simple refrigeration, preserving the fruit's quality and prolonging its shelf-life. All the treatments help to maintain the glucose and fructose content and the acidity, preserving the aroma and organoleptic characteristics.

Nutrients and Main Secondary Metabolites Characterizing Extracts and Essential Oil from Fruits of Ammodaucus leucotrichus Coss. & Dur. (Western Sahara).

The ethnobotany of the Sahrawi people considers various species of plants and crude drugs as food, cooking spices and traditional health remedies. From among these, the fruits of Ammodaucus leucotrichus Coss. & Dur. (Apiaceae), known as Saharan cumin, were chosen for our research. The present paper reports a proximate composition and mineral element analysis of various samples of A. leucotrichus fruits, collected during the balsamic period (full fruiting) from plants grown in Bir Lehlu (Western Sahara) and purchased in a local market (Tindouf). These analyses pointed out interesting nutritional values of the crude drug. Decoction and alcoholic extract, analyzed by HPLC-DAD, evidenced ammolactone-A and R-perillaldehyde as the two main isolated constituents, particularly in the ethanolic extracts (ammolactone-A, market sample: 51.71 ± 0.39 mg/g dry extract; wild sample: 111.60 ± 1.80 mg/g dry extract; R-perillaldehyde, market sample: 145.95 ± 0.35 mg/g dry extract; wild sample: 221.40 ± 0.30 mg/g dry extract). The essential oils, obtained through hydrodistillation, were characterized by GC-MS and evidenced R-perillaldehyde (market sample: 53.21 ± 1.52%; wild sample: 74.01 ± 1.75%) and limonene (market sample: 35.15 ± 1.68%; wild sample: 19.90 ± 1.86%) as the most abundant compounds. The R configuration of perillaldehyde was ascertained and a complete description of the 1H and 13C NMR spectra of ammolactone-A was performed.

Anticancer Activity of Aqueous Extracts from Asparagus officinalis L. Byproduct on Breast Cancer Cells.

Cultivation of asparagus (Asparagus officinalis L.; Asp) for food and medicinal use has taken place since the early Roman Empire. Today, Asp represents a worldwide diffuse perennial crop. Lower portions of the spears represent a food industry waste product that can be used to extract bioactive molecules. In this study, aqueous extracts derived from the non-edible portion of the plant (hard stem) were prepared and characterized for chemical content. Furthermore, the biocompatibility and bioactivity of Asp aqueous extracts were assessed in vitro on normal fibroblasts and on breast cancer cell lines. Results showed no interference with fibroblast viability, while a remarkable cytostatic concentration-dependent activity, with significant G1/S cell cycle arrest, was specifically observed in breast cancer cells without apoptosis induction. Asp extracts were also shown to significantly inhibit cell migration. Further analyses showed that Asp extracts were characterized by specific pro-oxidant activity against tumoral cells, and, importantly, that their combination with menadione resulted in a significant enhancement of oxidants production with respect to menadione alone in breast cancer cells but not in normal cells. This selectivity of action on tumoral cells, together with the easiness of their preparation, makes the aqueous Asp extracts very attractive for further investigation in breast cancer research, particularly to investigate their role as possible co-adjuvant agents of clinical drug therapies.

Antioxidant and Antiinflammatory Effects of Epilobium parviflorum, Melilotus officinalis and Cardiospermum halicacabum Plant Extracts in Macrophage and Microglial Cells.

BACKGROUND: We investigated the phenolic content characterizing different plant extracts from Epilobium parviflorum, Cardiospermum halicacabum, and Melilotus officinalis, their antioxidant, antiinflammatory effects, and their mechanism of action. METHODS: plant samples were macerated in 40% ethanol or hot/ cold glycerate and assessed for polyphenols content. The antioxidant activity was investigated by DPPH radical scavenging assay and H2DCFDA test in LPS-stimulated RAW264.7 macrophages and N9 microglial cells. MTS experiments and antiinflammatory properties verified cellular toxicity through NO assay. Interaction with A2A adenosine receptors was evaluated through binding assays using [3H]ZM241385 radioligand. RESULTS: Polyphenols were present in 40% ethanol plant extract, which at 0.1-10 µg/µL achieved good antioxidant effects, with a DPPH radical scavenging rate of about 90%. In LPS-stimulated cells, these plant extracts, at 1µg/µL, did not affect cell vitality, displayed significant inhibition of H2DCFDA and NO production, and inhibited ZM 241385 binding in CHO cells transfected with A2A receptors. RAW 264.7 and N9 cells presented a density of them quantified in 60 ± 9 and 45 ± 5 fmol/mg of protein, respectively. CONCLUSION: Epilobium parviflorum, Cardiospermum halicacabum, and Melilotus officinalis extracts may be considered a source of agents for treating disorders related to oxidative stress and inflammation.

Nutrient Composition and Antioxidant Performances of Bread-Making Products Enriched with Stinging Nettle (Urtica dioica) Leaves.

Stinging nettle (Urtica dioica) is an edible plant, well-known for its nutritional and nutraceutical properties. Stinging nettle leaves are typically rich in fibers, minerals and vitamins, as well as antioxidant compounds, i.e., polyphenols and carotenoids. Due to these reasons, since ancient times stinging nettle has been widely used in Italy as an ingredient in foods and beverages as a therapeutic agent. This work provides an investigation focused on bread enrichment with nettle leaves and the improvement of bread proximate composition in minerals, fibers and antioxidant compounds during product preparation. The comparison between plain and nettle enriched white bread shows a significant increase in fibers and nutrients, i.e., calcium and copper levels. Nettle enrichment also provides an increase in lutein and ß-carotene, as well as in total phenols and antioxidant activity. These last two nutritional elements are remarkably high in enriched bread and it has been found that phenolic concentration increases during breadmaking steps, from kneading to primary dough fermentation and from secondary fermentation of shaped loaves to baking.

Reconfigurable Filtering of Neuro-Spike Communications Using Synthetically Engineered Logic Circuits.

High-frequency firing activity can be induced either naturally in a healthy brain as a result of the processing of sensory stimuli or as an uncontrolled synchronous activity characterizing epileptic seizures. As part of this work, we investigate how logic circuits that are engineered in neurons can be used to design spike filters, attenuating high-frequency activity in a neuronal network that can be used to minimize the effects of neurodegenerative disorders such as epilepsy. We propose a reconfigurable filter design built from small neuronal networks that behave as digital logic circuits. We developed a mathematical framework to obtain a transfer function derived from a linearization process of the Hodgkin-Huxley model. Our results suggest that individual gates working as the output of the logic circuits can be used as a reconfigurable filtering technique. Also, as part of the analysis, the analytical model showed similar levels of attenuation in the frequency domain when compared to computational simulations by fine-tuning the synaptic weight. The proposed approach can potentially lead to precise and tunable treatments for neurological conditions that are inspired by communication theory.

Nanoparticulate Gels for Cutaneous Administration of Caffeic Acid.

Caffeic acid is a natural antioxidant, largely distributed in plant tissues and food sources, possessing anti-inflammatory, antimicrobial, and anticarcinogenic properties. The object of this investigation was the development of a formulation for caffeic acid cutaneous administration. To this aim, caffeic acid has been loaded in solid lipid nanoparticles by hot homogenization and ultrasonication, obtaining aqueous dispersions with high drug encapsulation efficiency and 200 nm mean dimension, as assessed by photon correlation spectroscopy. With the aim to improve the consistence of the aqueous nanodispersions, different types of polymers have been considered. Particularly, poloxamer 407 and hyaluronic acid gels containing caffeic acid have been produced and characterized by X-ray and rheological analyses. A Franz cell study enabled to select poloxamer 407, being able to better control caffeic acid diffusion. Thus, a nanoparticulate gel has been produced by addition of poloxamer 407 to nanoparticle dispersions. Notably, caffeic acid diffusion from nanoparticulate gel was eight-fold slower with respect to the aqueous solution. In addition, the spreadability of nanoparticulate gel was suitable for cutaneous administration. Finally, the antioxidant effect of caffeic acid loaded in nanoparticulate gel has been demonstrated by ex-vivo evaluation on human skin explants exposed to cigarette smoke, suggesting a protective role exerted by the nanoparticles.

Cu(II) coordination to His-containing linear peptides and related branched ones: Equalities and diversities.

The two branched peptides (AAHAWG)4-PWT2 and (HAWG)4-PWT2 where synthesized by mounting linear peptides on a cyclam-based scaffold (PWT2), provided with four maleimide chains, through a thio-Michael reaction. The purpose of this study was primarily to verify if the two branched ligands had a Cu(II) coordination behavior reproducing that of the single-chain peptides, namely AAHAWG-NH2, which bears an Amino Terminal Cu(II)- and Ni(II)-Binding (ATCUN) Motif, and HAWG-NH2, which presents a His residue as the N-terminal amino acid, in a wide pH range. The study of Cu(II) binding was performed by potentiometric, spectroscopic (UV-vis absorption, CD, fluorescence) and ESI-MS techniques. ATCUN-type ligands ((AAHAWG)4-PWT2 and AAHAWG-NH2) were confirmed to bind one Cu(II) per peptide fragment at both pH 7.4 and pH 9.0, with a [NH2, 2N-, NIm] coordination mode. On the other hand, the ligand HAWG-NH2 forms a [CuL2]2+ species at neutral pH, while, at pH 9, the formation of 1:2 Cu(II):ligand adducts is prevented by amidic nitrogen deprotonation and coordination, to give rise solely to 1:1 species. Conversely, Cu(II) binding to (HAWG)4-PWT2 resulted in the formation of 1:2 copper:peptide chain also at pH 9: hence, through the latter branched peptide we obtained, at alkaline pH, the stabilization of a specific Cu(II) coordination mode which results unachievable using the corresponding single-chain peptide. This behavior could be explained in terms of high local peptide concentration on the basis of the speciation of the Cu(II)/single-chain peptide systems.

Peptidomic Approach for the Identification of Peptides with Potential Antioxidant and Anti-Hyperthensive Effects Derived From Asparagus By-Products.

Asparagus waste represents products of great interest since many compounds with high biological value are located in the lower portion of the spears. The extraction of bioactive compounds from asparagus by-products is therefore crucial for the purpose of adding value to these by-products. In this paper, bioactive peptides from asparagus waste were extracted, digested, purified and identified. In particular, Alcalase® was chosen as the enzyme to use to obtain protein hydrolysate due to its low cost and, consequently, the possibility of implementing the method on a large scale. In order to simplify the peptide extract to reach better identification, the hydrolysate was fractionated by reversed-phase chromatography in 10 fractions. Two tests were carried out for antioxidant activity (ABTS-DPPH) and one for antihypertensive activity (ACE). Fractions with a higher bioactivity score were identified by peptidomics technologies and screened for bioactivity with the use of bioinformatics. For ACE-inhibitor activity, two peptides were synthetized, PDWFLLL and ASQSIWLPGWL, which provided an EC50 value of 1.76 µmol L-1 and 4.02 µmol L-1, respectively. For the antioxidant activity, by DPPH assay, MLLFPM exhibited the lowest EC50 value at 4.14 µmol L-1, followed by FIARNFLLGW and FAPVPFDF with EC50 values of 6.76 µmol L-1 and 10.01 µmol L-1, respectively. A validation of the five identified peptides was also carried out. The obtained results showed that peptides obtained from asparagus by-products are of interest for their biological activity and are suitable for being used as functional ingredients.

Chemical Composition of Essential Oils from Thymus vulgaris, Cymbopogon citratus, and Rosmarinus officinalis, and Their Effects on the HIV-1 Tat Protein Function.

New drugs would be beneficial to fight resistant HIV strains, in particular those capable of interfering with essential viral functions other than those targeted by highly active antiretroviral therapy drugs. Despite the central role played by Tat protein in HIV transcription, a search for vegetable extracts able to hamper this important viral function was never carried out. In this work, we evaluated the chemical composition and possible interference of essential oil from Thymus vulgaris, Cananga odorata, Cymbopogon citratus, and Rosmarinus officinalis with the Tat/TAR-RNA interaction and with Tat-induced HIV-1 LTR transcription. GC/MS Analysis demonstrated the biodiversity of herbal species translated into essential oils composed of different blends of terpenes. In all of them, 4 - 6 constituents represent from 81.63% to 95.19% of the total terpenes. Essential oils of Thymus vulgaris, Cymbopogon citratus, and Rosmarinus officinalis were active in interfering with Tat functions, encouraging further studies to identify single terpenes responsible for the antiviral activity. In view of the quite different composition of these essential oils, we concluded that their interference on Tat function depends on specific terpene or a characteristic blend.

Solid microparticles based on chitosan or methyl-ß-cyclodextrin: a first formulative approach to increase the nose-to-brain transport of deferoxamine mesylate.

We propose the formulation and characterization of solid microparticles as nasal drug delivery systems able to increase the nose-to-brain transport of deferoxamine mesylate (DFO), a neuroprotector unable to cross the blood brain barrier and inducing negative peripheral impacts. Spherical chitosan chloride and methyl-ß-cyclodextrin microparticles loaded with DFO (DCH and MCD, respectively) were obtained by spray drying. Their volume-surface diameters ranged from 1.77 ± 0.06 µm (DCH) to 3.47 ± 0.05 µm (MCD); the aerodynamic diameters were about 1.1 µm and their drug content was about 30%. In comparison with DCH, MCD enhanced the in vitro DFO permeation across lipophilic membranes, similarly as shown by ex vivo permeation studies across porcine nasal mucosa. Moreover, MCD were able to promote the DFO permeation across monolayers of PC 12 cells (neuron-like), but like DCH, it did not modify the DFO permeation pattern across Caco-2 monolayers (epithelial-like). Nasal administration to rats of 200 µg DFO encapsulated in the microparticles resulted in its uptake into the cerebrospinal fluid (CSF) with peak values ranging from 3.83 ± 0.68 µg/mL (DCH) to 14.37 ± 1.69 µg/mL (MCD) 30 min after insufflation of microparticles. No drug CSF uptake was detected after nasal administration of a DFO water solution. The DFO systemic absolute bioavailabilities obtained by DCH and MCD nasal administration were 6% and 15%, respectively. Chitosan chloride and methyl-ß-cyclodextrins appear therefore suitable to formulate solid microparticles able to promote the nose to brain uptake of DFO and to limit its systemic exposure.

GBA2-encoded ß-glucosidase activity is involved in the inflammatory response to Pseudomonas aeruginosa.

Current anti-inflammatory strategies for the treatment of pulmonary disease in cystic fibrosis (CF) are limited; thus, there is continued interest in identifying additional molecular targets for therapeutic intervention. Given the emerging role of sphingolipids (SLs) in various respiratory disorders, including CF, drugs that selectively target the enzymes associated with SL metabolism are under development. Miglustat, a well-characterized iminosugar-based inhibitor of ß-glucosidase 2 (GBA2), has shown promise in CF treatment because it reduces the inflammatory response to infection by P. aeruginosa and restores F508del-CFTR chloride channel activity. This study aimed to probe the molecular basis for the anti-inflammatory activity of miglustat by examining specifically the role of GBA2 following the infection of CF bronchial epithelial cells by P. aeruginosa. We also report the anti-inflammatory activity of another potent inhibitor of GBA2 activity, namely N-(5-adamantane-1-yl-methoxy)pentyl)-deoxynojirimycin (Genz-529648). In CF bronchial cells, inhibition of GBA2 by miglustat or Genz-529648 significantly reduced the induction of IL-8 mRNA levels and protein release following infection by P. aeruginosa. Hence, the present data demonstrate that the anti-inflammatory effects of miglustat and Genz-529648 are likely exerted through inhibition of GBA2.

Cochlear implants and drug delivery: In vitro evaluation of dexamethasone release.

Anti-inflammatory drugs can minimize the trauma and inflammation in the inner ear caused by cochlear implantation surgery. For this reason, much effort has recently been devoted to finding the best way to administer these anti-inflammatory drugs for a prolonged time and in a personalized dosage. One solution is constructing an electrode with a dispenser filled with anti-inflammatory drugs with a dosage adapted to suit each new cochlear implant user. The purpose of this study was to measure in vitro, by high-performance liquid chromatography-mass spectrometry assay, the amount of dexamethasone released in 78 days in a physiological solution by a filled dispenser. The drug release continued for more than 2 months in three different phases: (1) during the first 1-5 days, (2) within about 2 weeks, and (3) from about 3 weeks until the end of experiment. This release trend is in accordance with the 3 main phases of damage caused by the cochlear implantation: (1) insertion trauma within the first 2 days, (2) inflammation within 2 weeks, and finally (3) an intracochlear chronic fibrosis reaction. Future animal model studies should consider using this dispenser in order to establish its effectiveness in preventing damage caused by cochlear implantation.

Quantitative determination of zolmitriptan in rat blood and cerebrospinal fluid by reversed phase HPLC-ESI-MS/MS analysis: application to in vivo preclinical pharmacokinetic study.

A fast HPLC-ESI-MS/MS method has been developed and validated for the quantification of the potent and selective antimigraine zolmitriptan in rat blood and cerebrospinal fluid (CSF). The assay has been then applied for in vivo preclinical studies. The analytical determination has been used to obtain pharmacokinetics of zolmitriptan in the two biological matrices after its intravenous or nasal administration. Liquid-liquid extraction of zolmitriptan was performed from 100 µL rat blood samples in the presence of N(6)-cyclopentyladenosine (internal standard) with the employment of ethyl acetate. Calibration standards were prepared by using blood matrix and following the same liquid-liquid extraction procedure. CSF samples were analyzed without any pre-treatment steps and by using an external calibration method in pure water matrix. Chromatographic separation was performed under reversed phase and a gradient elution condition on a C18 packed column (100 × 2.0 mm, 2.5 µm particles diameter). The mobile phase was a mixture between acetonitrile, water and formic acid (0.1% v/v). The applied HPLC-MS/MS method allowed low limits of detection, as calculated from calibration curves, of 6.6 and 24.4 ng/mL for water matrix and rat blood extracts, respectively. Linearity of the calibration curves was established up to 5 µM (1.44 µg/mL), as well as good assay accuracy. The intravenous infusion of 20 µg zolmitriptan to male Sprague-Dawley rats produced blood concentrations ranging from 9.4±0.7 to 1.24±0.07 µg/mL within 10 h, with a terminal half-life of 3.4±0.2h. The nasal administration of a water suspension of 20 µg zolmitriptan produced blood concentrations ranging from 2.92±0.21 to 0.85±0.07 µg/mL within 6h. One hour after zolmitriptan intravenous infusion or nasal administration, its CSF concentrations were 0.0539±0.0016 and 0.0453±0.0012 µg/mL, respectively. This study determined the suitability of the herein proposed method to investigate the pharmacokinetics of zolmitriptan after its administration by means of novel formulations and, hence, to evaluate the efficacy of innovative nose-to-brain drug delivery in preclinical studies.

Oxaliplatin elicits mechanical and cold allodynia in rodents via TRPA1 receptor stimulation.

Platinum-based anticancer drugs cause neurotoxicity. In particular, oxaliplatin produces early-developing, painful, and cold-exacerbated paresthesias. However, the mechanism underlying these bothersome and dose-limiting adverse effects is unknown. We hypothesized that the transient receptor potential ankyrin 1 (TRPA1), a cation channel activated by oxidative stress and cold temperature, contributes to mechanical and cold hypersensitivity caused by oxaliplatin and cisplatin. Oxaliplatin and cisplatin evoked glutathione-sensitive relaxation, mediated by TRPA1 stimulation and the release of calcitonin gene-related peptide from sensory nerve terminals in isolated guinea pig pulmonary arteries. No calcium response was observed in cultured mouse dorsal root ganglion neurons or in naïve Chinese hamster ovary (CHO) cells exposed to oxaliplatin or cisplatin. However, oxaliplatin, and with lower potency, cisplatin, evoked a glutathione-sensitive calcium response in CHO cells expressing mouse TRPA1. One single administration of oxaliplatin produced mechanical and cold hyperalgesia in rats, an effect selectively abated by the TRPA1 antagonist HC-030031. Oxaliplatin administration caused mechanical and cold allodynia in mice. Both responses were absent in TRPA1-deficient mice. Administration of cisplatin evoked mechanical allodynia, an effect that was reduced in TRPA1-deficient mice. TRPA1 is therefore required for oxaliplatin-evoked mechanical and cold hypersensitivity, and contributes to cisplatin-evoked mechanical allodynia. Channel activation is most likely caused by glutathione-sensitive molecules, including reactive oxygen species and their byproducts, which are generated after tissue exposure to platinum-based drugs from cells surrounding nociceptive nerve terminals.

An insight into the radical thiol/yne coupling: the emergence of arylalkyne-tagged sugars for the direct photoinduced glycosylation of cysteine-containing peptides.

An explorative study of the Thiol-Yne Coupling (TYC) reaction has been carried out using an aliphatic (1-octyne) and an aromatic alkyne (phenylacetylene) and two alkanethiols (methyl thioglycolate and N-acetyl-L-cysteine methyl ester). The outcomes of the TYC reactions strongly depend on the experimental conditions (e.g., temperature, solvent, and alkyne/thiol ratio), but these can be properly adjusted to achieve selective production of either mono- or bis-coupling products. With respect to 1-octyne, phenylacetylene undergoes notably easier radical hydrothiolation, further showing a notably higher aptitude for monohydrothiolation exclusive of bis-hydrothiolation. The overall findings were exploited in glycosylation of cysteine derivatives as well as of cysteine-containing peptides. A sugar featuring an arylacetylene moiety gave rise to a true click-reaction, that is, glycosylation of the tripeptide glutathione in its native form, by means of virtually equimolar amounts of reagents. This reaction was successfully applied, under physiological conditions, to a cysteine-containing nonapeptide with marked advantages over the analogous Thiol-Ene Coupling (TEC) derivatization. A TYC/TEC sequence affording bis-armed cysteine derivatives through dual functionalization of an alkynyl sugar was additionally devised.

High peak capacity separations of peptides in reversed-phase gradient elution liquid chromatography on columns packed with porous shell particles.

The performance of 5 and 15 cm long columns packed with shell particles (Halo, AMT) is compared in gradient elution separations of the tryptic digests of myoglobin and bovine serum albumin. The influences of the temperature and the mobile phase flow rate on the column efficiency for two peptides are discussed. The influences of this flow rate, of the temperature, and of the gradient slopes on the peak capacities are also considered. Peak capacities in excess of 400 were achieved in 6h with the longer column. Peak capacities of 200 could be achieved in 30 min with the shorter column.

Comparison between the efficiencies of columns packed with fully and partially porous C18-bonded silica materials.

The chromatographic performance of a new brand of shell particles is compared to that of a conventional brand of totally porous silica particles having a similar size. The new material (Halo, Advanced Materials Technology, Wilmington, DE) is made of 2.7 microm particles that consist in a 1.7 microm solid core covered with a 0.5 microm thick shell of porous silica. The other material consists of the porous particles of a conventional 3 microm commercial silica-B material. These two columns have the same dimensions, 150 mm x 4.6mm. The reduced plate heights of two low molecular weight compounds, naphthalene and anthracene, two peptides (lys-bradykinin and bradykinin), and four proteins, insulin, lysozyme, beta-lactoglobulin, and bovine serum albumin were measured in a wide flow rate range and analyzed on the basis of the Van Deemter equation and of modern models for its terms. The Halo column provides a smaller axial diffusion coefficient B and a smaller eddy dispersion term A than the other column, a result consistent with its lower internal porosity (in(p)=0.19 versus 0.42) and with the narrower size distribution of its particles (sigma=5% versus 13%). The two columns have similar C terms for the two low molecular weight compounds and for the two peptides. However, the C term of the proteins that are not excluded is markedly lower on the column packed with the Halo particles than on the other column. A recent theoretical analysis of the mass transfer kinetics in shell particles predicts a C term for moderately retained proteins (3

Modeling of overloaded gradient elution of nociceptin/orphanin FQ in reversed-phase liquid chromatography.

The Reversed-phase (RP) gradient elution chromatography of nociceptin/orphanin FQ (N/OFQ), a neuropeptide with many biological effects, has been modeled under linear and non-linear conditions. In order to do this, the chromatographic behavior has been studied under both linear and nonliner conditions under isocratic mode at different mobile phase compositions--ranging from 16 to 19% (v/v) acetonitrile (ACN) in aqueous trifluoracetic acid (TFA) 0.1% (v/v)-on a C-8 column. Although the range of mobile phase compositions investigated was quite narrow, the retention factor of this relatively small polypeptide (N/OFQ is a heptadecapeptide) has been found to change by more than 400%. In these conditions, gradient operation resulted thus to be the optimum approach for non-linear elution. As the available amount of N/OFQ was extremely reduced (only a few milligrams), the adsorption isotherms of the peptide, at the different mobile phase compositions examined, have been measured through the so-called inverse method (IM) on a 5 cm long column. The adsorption data at different mobile phase compositions have been fitted to several models of adsorption. The dependence of the isotherm parameters on the mobile phase composition was modeled by using the linear solvent strength (LSS) model and a generalized Langmuir isotherm that includes the mobile phase composition dependence. The overloaded gradient separation of N/OFQ has been modeled by numerically solving the equilibrium-dispersive (ED) model of chromatography under a selected gradient elution mode, on the basis of the previously determined generalized Langmuir isotherm. The agreement between theoretical calculations and experimental overloaded band profiles appeared reasonably accurate.

Separation of peptides from myoglobin enzymatic digests by RPLC. Influence of the mobile-phase composition and the pressure on the retention and separation.

The influence of the mobile-phase composition and the pressure on the chromatographic separation of the peptides from the enzymatic digest of myoglobin was studied under linear conditions. The retention behavior of these tryptic peptides was measured under isocratic conditions with different mobile-phase compositions, ranging from 9 to 28% (v/v) acetonitrile in 0.1% (v/v) aqueous trifluoroacetic acid. The effect of the pressure was studied by analyzing the separation of the tryptic peptides under different average column pressures between 14 and 220 bar, at 13, 20, and 26% (v/v) acetonitrile. The differences between the partial molar volumes of these peptides in the stationary and mobile phases were derived from these results. All the measurements were performed on a 10-cm-long C18-bonded, end-capped monolithic column. The results obtained illustrate the highly complicated behavior of the complex peptide mixtures afforded by tryptic digestion. The capacity factors of the analyzed peptides do not depend linearly on the acetonitrile concentration but follow exactly a quadratic relationship. The adsorption changes of partial molar volumes are in good agreement with other literature data. The consequences of the influence of the average column pressure (hence of the flow rate) on the column phase ratio and on the retention factors of the peptides are discussed. The retention pattern of the complex mixture is affected by both the mobile-phase composition and the pressure, and the resolution of certain peptide pairs is so much affected by the pressure that inversions in the elution order of some pairs are observed.