Biomedical subjects

J Fiori

Publications and source records attributed to J Fiori.

13 recordsLinked to original sources

PubMed2006

Histone proteins determined in a human colon cancer by high-performance liquid chromatography and mass spectrometry.

The application of reversed-phase high-pressure liquid chromatography under gradient conditions and electrospray ion trap mass spectrometry (LC-ESI-MS) to the analysis of global modification levels of core histones is described. The optimised LC-ESI-MS method was applied for the first time to the characterisation of histones extracted from HT29, a human colon cancer cell line. Eight histones (H1-1, H1-2, H2A-1, H2A-2, H2B, H3-1, H3-2, H4) were separated on a C4 stationary phase with complete resolution, never reached in previous HPLC-MS methods, by using a gradient elution with the combined presence of heptafluorobutyric acid and formic acid as acidic modifiers in the mobile phase. Heptafluorobutyric acid was found to improve selectivity, whereas the presence of formic acid decreased ion suppression. Histones eluted from the column were detected with an ion trap mass spectrometer with an electrospray source. The peak averaged mass spectra were reconstructed by Mag Tran 1.0 software and the mass of the various isoforms of histones were derived. Method validation was conducted by performing the same sample analysis by coupling LC-ESI to a quadrupole-time-of-flight mass spectrometer (Q-TOF). The number of histone forms and their mass were found to differ not significantly from those obtained by ion trap mass spectrometer. Also the relative modifications abundance within the same histone type was found following the same trend as the two mass analysers. This method was then applied to the characterisation of changes in histone modification in HT29, never analysed by LC-MS before, treated with histone deacetylase inhibitors such as valproate and sodium butyrate, also used in preclinical trials as anticancer drugs. In particular, both the inhibitors produced a significant increase in H4 histone acetylated forms: 89% increase of the diacetyl dimethyl H4 form was observed with 1mM valproate supplementation, whereas 5 mM butyrate led to a 68% increase of the same form. Triacetyl monomethyl H4 (11,377 Da) and triacetyl dimethyl H4 (11,390 Da) were found only in cells treated with butyrate. Selective changes of H3 histone were detected with butyrate, in agreement with recently reported western blotting studies. Modifications in the H2A histone degree of acetylation were revealed by treatment of the cells with butyrate (H2A-1, H2A-2) and valproate (H2A-2). The results of the proposed methodology confirmed that inhibition of histone deacetylases caused histone hyperacetylation, responsible for decondensation and reorganization of interphase dynamic chromatin. This method resulted in selective and sensitive method to monitor variations in the acetylation and methylation state of histones after treatment of HT29 with inhibitors, and is therefore suitable for further application in new drug discovery for tumour therapy.

Acetylation↗

PubMed2006

Analysis of Amaryllidaceae alkaloids from Narcissus by GC-MS and capillary electrophoresis.

Amaryllidaceae are known as ornamental plants, furthermore some species of this family contain galanthamine, an acetylcholinesterase inhibitor approved for the treatment of Alzheimer's disease, and other alkaloids with interesting pharmacological activity. In the present work, the quali- and quantitative analysis of Amaryllidaceae-type alkaloids in the bulbs of Narcissus species is presented using different analytical approaches. Extracts of Narcissus pseudonarcissus cv. Carlton and Narcissus jonquilla Quail, were first examined by GC-MS using a Rtx-5 MS (programmed temperature) and the major alkaloids were identified. Together with galanthamine, high contents of haemanthamine, were found. Galanthamine was reliably quantified by GC-MS, whereas haemanthamine partly decomposed under the GC conditions, thus alternative analytical methods were investigated. Firstly, reversed-phase HPLC-ESI-MS was applied to identify and isolate at semipreparative levels haemanthamine. The compound was fully characterized by MS/MS and (1)H NMR and then used as a reference substance. The quantitation of both galanthamine and haemanthamine was then accomplished by capillary electrophoresis with spectrophotometric detection. A non-aqueous (NACE) approach was selected in order to use a running buffer fully compatible with samples in organic solvent. In particular, a mixture methanol-acetonitrile (75:25, v/v) containing ammonium acetate (90 mM) was used as a background electrolyte. The same analytical sample was subjected to GC-MS and NACE analysis; the different selectivity displayed by these techniques allowed different separation profiles that can be useful in phytochemical characterization of the extracts. The GC-MS and NACE methods were validated and applied to the quantitation of galanthamine (GC-MS and NACE) and haemanthamine (NACE) in bulbs of N. jonquilla.

Amaryllidaceae Alkaloids↗

PubMed2005

Liquid chromatography-tandem mass spectrometry for the identification of impurities in d-allethrin samples.

A previous GC/MS study highlighting the impurity profile of the synthetic pesticide d-allethrin is extended here to validate and confirm the impurities identity through the development of soft ionisation HPLC-MS methods. To accomplish this, we developed a reverse phase LC-MS analysis in gradient elution with two distinct soft ionisation techniques, the atmospheric pressure ionisation with electrospray source (API-ESI) and the chemical ionisation (APCI). A single quadrupole and an ion trap, which allowed the simultaneous determination of the molecular masses and structural information of the impurities by acquisition of collisionally induced (CID) product ions spectrum and in-source fragmentation, were employed as analysers. Single quadrupole and ion trap analysers resulted perfectly matching in the d-allethrin impurity fragmentation patterns. All the main impurities over 0.1% identified by GC/MS were confirmed. Results indicate that the proposed HPLC/MS method was found appropriate to confirm the presence of impurities such as chrysolactone, chloro allethrin derivatives, allethrolone and chrysanthemic acid, excluding their formation under GC/MS strong ionisation condition.

Allethrins↗

PubMed2005

HPLC-DAD and LC-ESI-MS analysis of doxycycline and related impurities in doxipan mix, a medicated premix for incorporation in medicated feedstuff.

HPLC-DAD and LC-ESI-MS methods have been developed for the analysis of doxycycline (DOX), including the identification of the related impurities metacycline (MTC) and 6-epidoxycycline (EDOX) and its determination in a medicated premix. The chromatographic separations have been performed on Luna C18 stationary phase and on Synergi (4 microm) Polar-RP 80A, using both acidic (pH 2.5) and basic (pH 8.0) mobile phases. The Synergi Polar-RP column, in combination with a mobile phase of oxalic acid (0.02 M; pH 2.5)-acetonitrile 82:18 (v/v), allowed the complete separation of MTC, EDOX and DOX. The same separation was also obtained using Luna C18 stationary phase with a pH 8 mobile phase. Application of a LC-ESI-MS system and MS/MS analysis, using both positive and negative polarity, allowed the peak identity to be confirmed. A method based on Luna C18 column and UV detection at 346 nm was validated for the determination of DOX in a medicated premix for incorporation in medicated feedstuff.

Animal Feed↗

PubMed2004

Stereoselective determination of allethrin by two-dimensional achiral/chiral liquid chromatography with ultraviolet/circular dichroism detection.

A two-dimensional achiral/chiral HPLC method with circular dichroism (CD) detection was optimized for the stereochemical resolution and determination of the elution order of the eight stereoisomers of synthetic allethrin. A monolithic silica HPLC column (Chromolith, Merck, 100 mm x 4.6 mm i.d.) was put orthogonally to an enantioselective OJ Daicel column (250 mm x 4.6 mm i.d.) by means of a switching valve. The resolution of cis and trans diastereoisomers on the silica column was obtained by using a mobile phase consisting of n-hexane:tert-butyl methyl ether (96:4) (v/v) at a flow rate of 1 ml min(-1). The cis and trans peaks were then switched to the enantioselective OJ column separately in two subsequent injections. The resolution of the four trans stereoisomers was accomplished by using n-hexane:tert-butyl methyl ether (90:10) (v/v), while the mobile phase composition for the four cis stereoisomers consisted of n-hexane:isopropanol (99.3:0.7) (v/v). The CD based detection system allowed the determination of the elution order on the basis of the CD signals of the single stereoisomers, together with the injection of pure stereoisomers. Under the final conditions, the validated method was applied to the determination of stereoisomeric composition and absolute configuration of the prevailing stereoisomers of real samples, i.e. commercial batches of different sources of d-allethrin.

Allethrins↗

PubMed2004

Photostability studies on nicardipine-cyclodextrin complexes by capillary electrophoresis.

Nicardipine (NC)-cyclodextrin solid systems were prepared in equimolar ratios and their photostability in aqueous solution under exposure to UV(A)-UV(B) radiations was evaluated. The photodegradation process was monitored by a capillary electrophoresis (CE) method able to provide the enantioresolution of the rac-nicardipine. Enantioresolution was achieved using the mixture 3.0% sulfate-beta-cyclodextrin (SbetaCD) and 2.0% heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin (TMbetaCD) as chiral selector in 20mM triethanolammonium phosphate solution (pH 3.0). The photostability studies were carried out on inclusion complexes of rac-nicardipine with alpha-cyclodextrin (alphaCD), beta-cyclodextrin (betaCD), gamma-cyclodextrin (gammaCD), hydroxypropyl-alpha-cyclodextrin (HPalphaCD), hydroxypropyl-beta-cyclodextrin (HPbetaCD), hydroxypropyl-gamma-cyclodextrin (HPgammaCD), (2-hydroxyethyl)-beta-cyclodextrin (HEbetaCD) and methyl-beta-cyclodextrin (MbetaCD). A photoprotective effect was observed by betaCD, HPalphaCD, HEbetaCD, whereas gammaCD, MbetaCD, HPbetaCD and HPgammaCD did not affect the nicardipine photostability. Conversely, alphaCD was found to favour the drug photodegradation. Evidences for CDs-mediated stereoselective photodegradation of rac-nicardipine were observed only for the beta-CD complex. In this case, two distinct photodegradation profiles, with two different kinetic constants (k), were observed for the nicardipine enantiomers.

Biotransformation↗

PubMed2003

Photostability studies on the furosemide-triamterene drug association.

The photostability of the diuretic drugs triamterene and furosemide, individually and combined, was evaluated. Spectrophotometric, spectrofluorimetric and chromatographic (HPLC) methods were applied to monitor the drug photodegradation. Furosemide was confirmed to be highly photolable in both pH 7.4 solution and methanol. Differently triamterene proved to be highly fluorescent (emission quantum yield: 0.9 in methanol and 0.8 in pH 7.4 solution), but essentially photostable (photochemical reaction quantum yield: congruent with 5 x 10(-4)) under exposure at 365 and 313 nm radiations. When the combined drugs in pH 7.4 solutions were exposed to 365 nm radiations a significant photoprotective effect of triamterene on furosemide was observed. The photoreactivity of the drugs was exploited to develop an HPLC method involving a post-column on-line photochemical derivatization useful to confirm the analyte identity in a commercial dosage form (tablets). The commercial product, containing the combined drugs, proved to be photostable also after long (65 h) light exposure.

Chromatography, High Pressure Liquid↗

PubMed2003

Analysis of glycolytic enzyme co-localization in Drosophila flight muscle.

In Drosophila flight muscles, glycolytic enzymes are co-localized along sarcomeres at M-lines and Z-discs and co-localization is required for normal flight. We have extended our analysis of this phenomenon to include a set of six glycolytic enzymes that catalyze consecutive reactions along the glycolytic pathway: aldolase, glycerol-3-phosphate dehydrogenase (GPDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), triose phosphate isomerase, phosphoglycerate kinase and phosphoglycerol mutase (PGLYM). Each of these enzymes has an identical pattern of localization. In mutants null for GPDH, localization of none of the other enzymes occurs and therefore is interdependent. In optimally fixed preparations of myofibrils, accumulation of the enzymes at M-lines is much greater than at Z-discs. However, localization at M-lines is more labile, as shown by loss of localization when fixation is delayed. We have begun to analyze the protein-protein interaction involved in glycolytic enzyme co-localization using the yeast two-hybrid system. We have identified two pair-wise interactions. One is between GPDH and GAPDH and another is between GPDH and PGLYM.

Animals↗

PubMed2003

Photomutagenic properties of terfenadine as revealed by a stepwise photostability, phototoxicity and photomutagenicity testing approach.

Administration of the second-generation antihistamine, terfenadine, is sometimes associated with photosensitivity and other skin reactions. To obtain information on its photoreactivity, we used a stepwise experimental approach involving tests for photostability, phototoxicity (PT) (mouse fibroblast cell line [3T3] neutral red uptake [NRU] test) and photomutagenicity (with standard Ames salmonella tester strains TA98, TA100 and TA102). Terfenadine was not phototoxic to cultured mammalian cells under the conditions used (i.e. 5000/161 mJ cm(-2) UVA-UVB). Natural sunlight and UV radiations caused considerable drug decomposition and formation of several photoproducts. Addition of the irradiated terfenadine solution (i.e. a mixture of photoproducts) to the tester did not significantly increase background mutation frequency. Irradiation of terfenadine coplated with the TA102 strain induced a clear-cut photomutagenic response, the magnitude of which was dependent upon the precursor compound concentration and the UV dose (212/7 to 339/11 mJ cm(-2) UVA-UVB). These findings demonstrate that in vitro terfenadine is photomutagenic in absence of PT. Further in vitro and in vivo studies are therefore needed to provide an adequate safety assessment of the photochemical genotoxicity--carcinogenicity potential of terfenadine. In the meantime, patients should be advised to avoid excessive exposure to sunlight.

3T3 Cells↗

PubMed2002

Cytotoxic and cytostatic effects induced by 4-hydroxynonenal in human osteosarcoma cells.

Several studies point to the existence of an inverse correlation between cellular lipid peroxidation and both cell proliferation and neoplastic transformation. Furthermore, numerous results demonstrate that lipid peroxidation products affect central biochemical pathways and intracellular signalling at physiological concentrations. 4-Hydroxynonenal (HNE) is one of the most active products of lipid peroxidation. This work has focused on the evaluation of HNE nuclear content, so far never directly measured, by electrospray-ionization-mass-spectrometry (ESI/MS) and on the correlation between its concentration and the induced effects after exogenous administration. In a human osteosarcoma cell line (SaOS2), HNE exhibited an early cytotoxic effect characterized by apoptosis, cytostatic and differentiating effects characterized by slow growth, increase in alkaline phosphatase (ALP), and alpha5 integrin subunit content with decrease in tumorigenicity.

Aldehydes↗

PubMed2002

Photodegradation studies on lacidipine in solution: basic experiments with a cis-trans reversible photoequilibrium under UV-A radiation exposure.

The photostability of Lacidipine, a dihydropyridine drug used in the treatment of mild and moderate hypertension, was studied in solutions exposed to UV-A radiations. The effects of the solvent (ethanol, acetone, dichloromethane), drug concentration and radiation wavelength on the drug photostability were evaluated. Lacidipine and its photoproducts were separated by a selective liquid chromatographic (HPLC) method, under normal phase conditions (CN-column), using n-hexane:ethanol 97:3 (v/v) as mobile phase, at a flow rate of 2.0 ml/min. The main photodegradation products were isolated and characterised and a photodegradation pathway was proposed for Lacidipine in solution. The cis-isomer and a photocyclic isomer proved to be the main photodegradation products.

Calcium Channel Blockers↗

PubMed2002

Determination of triclosan in personal health care products by liquid chromatography (HPLC).

An isocratic reversed-phase liquid chromatographic (HPLC) method is proposed for the practical and reliable determination of triclosan, an antimicrobic agent incorporated into a variety of personal heath care products. Chromatographic separations were performed on a C-18 column using acetonitrile-TEA phosphate (70 mM; pH 3.5) 55:45 (v/v) as mobile phase and UV detection at 230 and 280 nm. The selectivity of the method was assured by the on-line photodiode array detector. The identity of the triclosan peak was also confirmed by HPLC MS. The method was successfully applied to the determination of triclosan in commercially available health care products (deodorant stick, dentifrice gel, mouthrinse, toothpaste and handwash). All the products displayed triclosan concentrations in compliance with the EEC directive (< or = 0.3%,).

Anti-Infective Agents↗

PubMed2001

Chromatographic (GC-MS, HPLC) and virological evaluations of Salvia sclarea infected by BBWV-I.

Salvia sclarea cultivated at the Herb Garden of Casola-Valsenio (Emilia-Romagna region, Italy) has been found for the first time naturally infected by broad bean wilt fabavirus, serotype I (BBWV-I). Symptomatic plants showed malformed leaves, with chlorotic mosaic followed by yellowing and stunting. BBWV-I was identified by applying virological tests: mechanical inoculations on herbaceous plants, electron microscopy, DAS-ELISA and PAS-ELISA. The essential oil obtained from BBWV-infected material corresponded to 2/3 the quantity of that from healthy material. The GC-MS and HPLC analyses of these oils afforded a comparative analytical profile of the two plant materials attributed to BBWV-I infection. The oils from infected materials showed higher percentages of sesquiterpene hydrocarbons (e.g. germacrene D and beta-caryophyllene), monoterpene alcohols (e.g. alpha-terpineol) and diterpenoids (mainly sclareol). In contrast, lower levels of monoterpene hydrocarbons (e.g. myrcene, limonene and the two ocimene isomers) and the principal components (linalyl acetate and linalool) were observed.

Chromatography, High Pressure Liquid↗