Francesco Botrè

Honeybees and their products as potential bioindicators of heavy metals contamination

The concentrations of three representative heavy metals(cadmium, chromium and lead) were measured by atomic absorptionspectroscopy in honeybees and in apiarys products (honey,pollen, propolis, and wax). Samples were collected from fivedifferent sampling points: four from areas surrounding the cityof Rome, and the fifth in the city center which receives intensevehicular traffic. All apiaries employed for this study werespecifically constructed without any metal part in order toavoid the risk of contamination of the assayed materials.Sample collection was conducted over a 3-month period (6samplings for honey and pollen, 3 sampling for propolis and wax,2 samplings for honeybees, all of which were collected in duplicate). Experimental data revealed, in general,statistically significant differences between the backgroundlevels of heavy metals recorded from the reference sites and thelevels measured in the site located in the center of the city ofRome.These results indicate that honeybees and, to a lesser extent,some of their products (pollen, propolis, wax, but not honey),can be considered representative bioindicators of environmentalpollution.

Ascorbic acid in exotic fruits: a liquid chromatographic investigation

Abstract The levels of ascorbic acid (AA) have been measured by means of an HPLC method in 11 different exotic fruits (avocado pear, babaco, feijoa, grapefruit, kiwi, kumquat, litchi, mango, papaya, passion fruit, pineapple) and, for comparative purposes, in two citrus fruits (lemon and orange). They were measured in the exotic fruits at two different stages of ripening: (i) immediately after purchasing from a local fresh fruit market, and (ii) after a one-week period of artificial ripening. The results show that all tropical fruits contain relatively high levels of AA (varying between 20 and 90 mg/100g), with the exceptions of avocado pear and feijoa (whose AA levels are markedly affected by oxidation processes). Moreover, the results show that there is a remarkable loss of AA content (usually 30–40%) after the one-week period of artificial ripening, in all the different tropical fruits considered. They seem to indicate that the process of artificial ripening, which is usually carried out during the short-term storage of exotic fruits, can affect the nutritional value of this kind of food as far as the concentration of the reduced form of vitamin C is concerned.

Multifunctional au nanoparticle dendrimer-based surface plasmon resonance biosensor and its application for improved insulin detection.

Bifunctional hydroxyl/thiol-functionalized fourth-generation polyamidoamine dendrimer (G4-PAMAM)-encapsulated Au nanoparticle (NP) was synthesized and immobilized on a mixed self-assembled monolayer (SAM)-modified gold surface. This modified surface was resistant to nonspecific adsorption of proteins having a wide range of molecular weight and isoelectric points. Part of the dendrimer thiol groups were converted to hydrazide functionalities providing an activated surface available to subsequently immobilize the receptor for developing a sensor surface to immunoaffinity reaction. Herein, the surface plasmon resonance (SPR) detection of insulin was obtained by means of a competitive immunoassay principle. The resulting Au NP dendrimer-modified surface provided an assay with high stability, significantly enhanced sensitivity, and a detection limit for analyzing insulin of 0.5 pM. The SPR detection of insulin was amplified due to the changes in the dielectric properties of the matrixes, occurring upon the biorecognition processes on the sensor surface, through the coupling of the localized plasmon of the NPs with the surface plasmon wave. The developed sensor chip was used to analyze insulin in human serum samples from healthy and diabetic patients. The results showed good correlation to the reference method. The specificity and the improved sensitivity of this biosensing platform could have significant implications for the detection of a wide range of molecules and biomarkers in complex biological media.

Determination of clenbuterol in human urine by GC–MS–MS–MS: confirmation analysis in antidoping control

This work presents a GC-MS-MS-MS method for the direct determination of clenbuterol in human urine. The method comprises a pretreatment procedure and the instrumental analysis of the derivatives performed by GC-MS(3) (ion trap) with electron impact ionization. The GC-MS(3) analysis allows isolation and characterization of specific fragments from the original (MS(1)) molecular structure, and in particular, those fragments originating from the precursor ion cluster (m/z=335-337) characteristic of clenbuterol. The MS(2) product fragment m/z=300 is in turn used as a further precursor fragment giving rise to a MS(3) spectrum specific for clenbuterol. MS(4) fragmentation spectra were also investigated. However, further fragmentation of MS(3) product ions does not lead to functional MS(4) spectra nor to any significant increase in the signal-to-noise ratio. The sensitivity limit of the MS(3) technique is lower than 0.2 microg/l, with a linear range between 0.5 and 5 microg/l, thus matching the basic requirements for antidoping analysis according to the guidelines of the International Olympic Committee. Due to its overall analytical performance, the method is presently being evaluated as a confirmation protocol to be followed to detect illicit clenbuterol administration to the athletes, and compared with reference GC-MS and GC-MS-MS techniques.

The abuse of diuretics as performance-enhancing drugs and masking agents in sport doping: pharmacology, toxicology and analysis

Diuretics are drugs that increase the rate of urine flow and sodium excretion to adjust the volume and composition of body fluids. There are several major categories of this drug class and the compounds vary greatly in structure, physicochemical properties, effects on urinary composition and renal haemodynamics, and site and mechanism of action. Diuretics are often abused by athletes to excrete water for rapid weight loss and to mask the presence of other banned substances. Because of their abuse by athletes, diuretics have been included on The World Anti‐Doping Agencys (WADA) list of prohibited substances; the use of diuretics is banned both in competition and out of competition and diuretics are routinely screened for by anti‐doping laboratories. This review provides an overview of the pharmacology and toxicology of diuretics and discusses their application in sports. The most common analytical strategies currently followed by the anti‐doping laboratories accredited by the WADA are discussed along with the challenges laboratories face for the analysis of this diverse class of drugs.

Acid phosphatase/glucose oxidase-based biosensors for the determination of pesticides

Abstract This work presents new amperometric bienzymatic bioelectrodes for the determination of organophosphorus and carbamic acid type pesticides. Two different kinds of bienzymatic bioelectrodes are presented: a classical bienzymatic electrode, obtained by physicochemical immobilization of purified acid phosphatase (AP) and glucose oxidase (GOD) on the tip of an amperometric H2O2 electrode; and a hybrid biosensor, in which AP has been employed in the form of a thin layer of potato (Solanum tuberosum) tissue, endowed with a high content of enzyme activity. Both the biosensors can selectively detect glucose-6-phosphate (G6P), in the 5.0 × 10−5 −1.2 × 10−3M concentration range. Pesticides are detected, thanks to their high inhibition power towards AP, evaluated by adding the sample stepwise to a buffered solution of G6P, and recording the corresponding current change. The detection limit is therefore a function of the type of pesticide, but it can be as low as 1 μg 1−1 in the case of organophosphorus compounds. The detection limit is generally higher for carbamates, as a consequence of their weaker inhibition power towards acid phosphatase. Both bioelectrodes presented comparable values of the main physicochemical and analytical parameters evaluated for assessing their overall performance; nonetheless the plant tissue based bioelectrode exhibited a longer shelf life and a better reliability of the amperometric results.

Detection of beta-blockers in human urine by GC-MS-MS-EI: perspectives for the antidoping control.

We have developed a general method for the detection of beta-blockers and/or of their metabolites in human urine. The method comprises a pretreatment procedure (enzymatic hydrolysis, liquid/liquid extraction and derivatization by pentafluoropropionic anhydride, PFPA), carried out on an initial aliquot of 2.5-5.0 ml of urine, and the instrumental analysis of the derivatives, performed by GC-MS-MS (ion trap) with electronic impact ionization (EI). The GC-MS-MS analysis allows to isolate and to characterize specific fragments of the original molecular structure, and particularly the fragments originating from parent ion clusters specific for all beta blocking drugs, giving rise to m/z = 366 and 202 ions respectively. MS-MS analysis of the parent ion allows checking for the presence of the above-mentioned peaks in the GC-MS chromatogram. The proposed method is capable of detecting a great variety of known (and possibly also of newly synthesized) beta-blockers, with an average sensitivity limit of 20 ng/ml of drug/metabolite in urine. The method is presently being evaluated as a general screening protocol to be followed by an antidoping laboratory to detect illicit beta-blockers administration to the athletes.

A gas chromatography/mass spectrometry method for the determination of sildenafil, vardenafil and tadalafil and their metabolites in human urine

Sildenafil (SDF), vardenafil (VDF) and tadalafil (TDF) are phosphodiesterase type 5 enzyme inhibitors (PDE5Is), used in the treatment of erectile disorders and to improve breathing efficiency in pulmonary hypertension. The increasing incidence of their use among young athletes has drawn the attention of the anti-doping authorities to the possible abuse of PDE5Is by athletes due to their pharmacological activities. This paper describes a method for the determination in urine of PDE5Is and their metabolites by gas chromatography/mass spectrometry (GC/MS) after liquid/liquid extraction of the analytes from urine and derivatisation to obtain trimethylsilyl derivatives. The metabolic profile was studied on real samples collected from subjects taking PDE5Is (Viagra, Levitra or Cialis); the main urinary metabolites were identified and their MS fragmentation characterized. The sample pre-treatment and GC/MS conditions for the detection of the metabolites have been optimised. A method for their preliminary screening and subsequent confirmation is described that takes into account the general requirements of a routine doping analysis to be used for the screening of large numbers of samples. The main metabolites identified can be included in a general purpose screening method and all the metabolites in a more specific confirmation method. The method developed has been applied for the screening of PDE5Is in 5000 urine samples. Based on the obtained results, the proposed method appears to be of practical use in analytical and forensic toxicology, including doping analysis.

Plant tissue electrode for the determination of atrazine

Abstract This work presents a new method for the simple and inexpensive determination of atrazine. The method is based on the use of a novel, partially disposable, plant tissue bioelectrode, which is sensitive to a variety of mono- and polyphenols. The biosensor is obtained by coupling a thin slice of potato ( Solanum tuberosum ) tissue, which contains high levels of the enzyme polyphenoloxidase (PPO), to a commercial O 2 -selective Clark electrode. The concentration of atrazine in aqueous samples can be determined thanks to its inhibitory power toward the catalytic activity of PPO. The low cost of this device and its good analytical performance suggest its application in the field of environmental analysis, especially in the continuous monitoring of atrazine in risk areas.

Lichen Usnea barbata as biomonitor of airborne elements deposition in the Province of Tierra del Fuego (southern Patagonia, Argentina).

Lichen Usnea barbata was tested as a possible biomonitor of atmospheric deposition in a supposedly pristine environment Tierra del Fuego (Argentina). Lichen samples were collected in 2005 and again in 2006 in 71 sites covering almost the entire region. The aim of the study was to evaluate the bioaccumulation of 26 elements in order to define the background levels in the region. The quantification was carried out by the sector field inductively coupled plasma mass spectrometry. No relevant temporal accumulation patterns between 2005 and 2006 sampling campaigns were observed. Then, the results were submitted to multivariate statistical analysis (cluster and principal component analyses). Cluster analysis produced a dendrogram where the 71 sites were divided into four clusters at (Dlink/Dmax)100<30. The areas and the elements were correlated according to the element concentrations by principal component analysis. Four significant components that accounted for 67% were obtained. Cluster 1 was mainly composed of sites of Ushuaia-Road 3 (E area) and it was characterized by high levels of Cd, Co, Ni, Pb, Sb, and W in lichens. The present study has revealed the good capacity of U. barbata to reflect the baseline levels of elements in the environment at a regional scale level. The presence of certain level of elements in lichens agrees with the hypothesis that Tierra del Fuego is not a relatively pristine environment as occasionally supposed. However, when comparing our results with other countries, Tierra del Fuego lichens have a very low content of the measured elements.