Andrea Raffaelli

Non-covalent complexes between DNA-binding drugs and double-stranded deoxyoligonucleotides: a study by ionspray mass spectrometry

The non-covalent complexes between some DNA-binding drugs and duplex oligodeoxynucleotides were studied by ionspray mass spectrometry, with the aim of evaluating the suitability of this technique to screen rapidly a series of drugs exerting their activity through non-covalent binding to specific base sequences of DNA. Two classes of drugs were considered, distamycins (which show affinity for the minor groove of DNA) and anthracyclines (which interact through intercalation between bases). For the former, d(CGCGAATTCGCG)2 was chosen as the model oligodeoxynucleotide. Following optimization of sample preparation and instrumental conditions, the complexes of different distamycins were observed; depending on the ligand considered, 1:1 or 2:1 complexes were formed preferentially. A semi-quantitative evaluation of the relative affinities was made by measuring the ratio of the complexes signals to those of the duplex, and also by competitive binding with equimolar amounts of distamycin. For anthracyclines, the daunorubicin-d(CGATCG)2 complex was chosen as the model for a preliminary mass spectrometric study; however, the signals of the duplex and the complex were very low compared with the monomer signal. Since the complex was known to be stable in solution, this was ascribed to gas-phase instability, probably caused by electrostatic repulsion between negatively charged phosphate groups.

Tissue Distribution and Cardiac Metabolism of 3-Iodothyronamine

3-iodothyronamine (T1AM) is a novel relative of thyroid hormone, able to interact with specific G protein-coupled receptors, known as trace amine-associated receptors. Significant functional effects are produced by exogenous T1AM, including a negative inotropic and chronotropic effect in cardiac preparations. This work was aimed at estimating endogenous T1AM concentration in different tissues and determining its cardiac metabolism. A novel HPLC tandem mass spectrometry assay was developed, allowing detection of T1AM, thyronamine, 3-iodothyroacetic acid, and thyroacetic acid. T1AM was detected in rat serum, at the concentration of 0.3±0.03 pmol/ml, and in all tested organs (heart, liver, kidney, skeletal muscle, stomach, lung, and brain), at concentrations significantly higher than the serum concentration, ranging from 5.6±1.5 pmol/g in lung to 92.9±28.5 pmol/g in liver. T1AM was also identified for the first time in human blood. In H9c2 cardiomyocytes and isolated perfused rat hearts, significant Na+-dependent uptake of exogenous T1AM was observed, and at the steady state total cellular or tissue T1AM concentration exceeded extracellular concentration by more than 20-fold. In both preparations T1AM underwent oxidative deamination to 3-iodothyroacetic acid. T1AM deamination was inhibited by iproniazid but not pargyline or semicarbazide, suggesting the involvement of both monoamine oxidase and semicarbazide-sensitive amine oxidase. Thyronamine and thyroacetic acid were not detected in heart. Finally, evidence of T1AM production was observed in cardiomyocytes exposed to exogenous thyroid hormone, although the activity of this pathway was very low.

Mass spectrometric characterization of flavonoids in extracts from Passiflora incarnata

Abstract Passiflora incarnata is a medicinal plant widely used for its sedative properties. The type of active compounds responsible for the sedative effects is not known yet, despite the fact that flavonoids are its most abundant active compounds. We present here a mass spectrometry study by ionspray ionization of extracts from Passiflora incarnata . The use of on-line HPLC–tandem mass spectrometry allowed the characterization of most of the flavonoids present. The interpretation of [M−H] − product ion spectra permitted the identification of several isomeric flavonoids containing different sugar moieties on the same aglycone. Some minor components were partially characterized. Their possible structures have been postulated on the basis of their [M−H] − product ion spectra.

Virtual freezing of the hemiacetal–aldehyde equilibrium of the aglycones of oleuropein and ligstroside present in olive oils from Carolea and Coratina cultivars by ionspray ionization tandem mass spectrometry

The composition of the equilibrium between aldehydes and hemiacetals was evaluated by ionspray ionization tandem mass spectrometry. The methanolic extracts of virgin olive oil from Carolea and Coratina cultivars contain a complex mixture of hydroxytyrosol (3) and tyrosol (4) derivatives (5, 6) characterized by the presence of aldehyde moieties in equilibrium with their hydrated form (8) or with the corresponding methanol hemiacetals 9 and 10. The equilibrium was frozen by droplet evaporation, thus allowing the structure determination of each component. The formation of the decarboxylated species 11-14, reported previously, was not observed.

Direct Identification of Phenolic Glucosides from Olive Leaf Extracts by Atmospheric Pressure Ionization Tandem Mass Spectrometry

Pneumatically assisted electrospray (or ionspray) coupled with liquid chromatography was applied to the identification of the phenolic glucoside content of olive leaf directly from the crude extracts. The mass spectra of the positive ions provide insights into the composition of the phenolic constituents. Oleuropein, ligstroside and a disaccharide containing the hydroxytyrosol moiety were found in olive leaf of Olea europea L. cv. Cassanese and their structures were thoroughly determined by tandem mass spectrometry.

(Z)-9-tricosene identified in rectal gland extracts of Bactrocera oleae males: first evidence of a male-produced female attractant in olive fruit fly.

It is well-known that Bactrocera oleae (olive fruit fly) females attract conspecific males by using 1,7-dioxaspiro[5,5]undecane (1) as the main component of their sex pheromone, and that 1 is produced in the female rectal gland. Although some authors have claimed that B. oleae males also attract females, to date no male-produced female attractants have been found in this species. In this paper, we report the first identification of a substance unique to males and able to attract females. The findings of the study include the following: (1) females responded in a bioassay to hexane extracts obtained from rectal glands of 15-day-old B. oleae males, (2) the presence of (Z)-9-tricosene (2) was consistently and unambiguously identified in these extracts using gas chromatography (GC) and GC-mass spectrometry methods, (3) in preliminary bioactivity tests, low doses (equivalent to a few males) of chemically and stereoisomerically pure synthetic (Z)-9-tricosene (2) attracted olive fruit fly females. Interestingly, compound 2, commonly called muscalure, is also a well-known component of the house fly (Musca domestica) sex pheromone.

Characterization of cassanese olive cultivar through the identification of new trace components by ionspray tandem mass spectrometry

Pneumatically assisted electrospray (ionspray) coupled with liquid chromatography was applied in the identification of antioxidants present in traces in the leaves of Olea europea L. cv. Cassanese, a typical olive tree from the northeastern part of Calabria region in Italy. The structures of these potential biomarkers were evaluated with reference to established mass spectrometric rules or, in one case, by matching the tandem mass spectrum of the unknown with that of an authentic sample obtained by synthesis. Copyright

Rhodium catalyzed deuteroformylation of styrene: (E)- and (Z)-β-deuterostyrene and β,β-dideuterostyrene formation via selective β-hydride elimination from the branched alkylrhodium intermediate

Deuteroformylation of styrene in the presence of Rh4(CO)12 as a catalytic precursor was carried out at 160 atm of CO and D2 1/1 at two temperatures (20 and 90°C) and for times yielding partial or complete conversion. Compounds recovered from the mixture produced by reaction and partial conversion at 90°C include unlabeled styrene, (E)- and (Z)-β-deuterostyrene, C6H5CHCHD, and β,β-dideuterostyrene, C6H5CHCD2, whereas at room temperature the styrene does not take up deuterium. These results indicate that under hydroformylation conditions the branched alkylrhodium intermediate, which affords the branched aldehyde, in part dissociates into rhodium hydride and deuterated olefin. By contrast the linear alkyl intermediate does not dissociate under the same conditions, but instead yields almost completely the corresponding aldehyde.

Characterization of psoralen-oleic acid cycloadducts and their possible involvement in membrane photodamage.

By UVA irradiation of an ethanol solution of psoralen and oleic acid, four main photoproducts have been isolated and characterized: two have cis,cis structure; the other two are trans,cis. The same adducts have been isolated from the photoreaction of psoralen with beta-oleoyl-gamma-stearoyl-1-alpha-phosphatidylcholine followed by enzymatic hydrolysis with phospholipase A2. The four isomers stimulate protein kinase C to almost the same extent.

Recent advances in the assessment of the ratios of cortisol to cortisone and of some of their metabolites in urine by LC-MS-MS.

A previously reported method for the assessment of the ratio of tetrahydrocortisol (THF) + allo-tetrahydrocortisol (A-THF) to tetrahydrocortisone (THE) by HPLC-MS-MS has been significantly improved, in order to increase either ruggedness and reliability. That was achieved by the introduction of an on-line sample cleanup stage, which made use of a perfusion column as a solid phase microextraction (SPE) cartridge. The set of analytes was expanded, by introducing cortisol and cortisone, whose ratio supply additional diagnostic information. The response factors of both THF and A-THF has been checked, resulting almost identical, as well as the influence of the matrix on the calibration curves which, although different for water and urine, had similar effect on the ratios of interest. As a consequence, the calibration solutions can be prepared in pure water. The influence of several different storage procedures has also been tested, resulting in no substantial effect on the final result. Finally, the improved method has been used to run real samples from healthy volunteers, with satisfactory results.