Luca Amendola

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.

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.

Analysis of organophosphorus pesticides by gas chromatography–mass spectrometry with negative chemical ionization: a study on the ionization conditions

Abstract The present paper is aimed to study the optimal ionization conditions of 19 organophosphorous pesticides to be analyzed by gas chromatography (GC) with mass spectrometric (MS) detection in negative chemical ionization (NCI). Isobutane, methane, ammonia in methane, and pure ammonia were used as ionizing gases. The effect of the source temperature on the mass spectrum was evaluated for each pesticide considered in this study and for each ionizing gas. The influence of the electron energy, of the emission intensity and of the gas pressure was also studied for ammonia and ammonia in methane. The MS profiles have shown that NCI fragmentation induced by methane, isobutane and ammonia, generally leads to fragmentation by electron capture. Furthermore, significant differences in the mass spectra of several pesticides were recorded by changing the ionizing gas. Finally, the use of pure ammonia leads to a marked reduction of the background noise, with a parallel improvement of the overall sensitivity of the method, with values of the limit of detection (LOD) lower ( −1 ) than those obtained by methane or isobutane.

Halocarbons in Aqueous matrices from the Rennick Glacier and the Ross Sea (Antarctica)

Aqueous matrices from Antarctica were analysed for three volatile chlorinated hydrocarbons (VCHCs): tetrachloromethane (CCl4), trichloroethylene (C2HCl3) and tetrachloroethylene (C2Cl4). The matrices analysed were snow from Rennick Nèvè and Rennick Glacier sampled during the Italian Expeditions of 1995/96 and 1996/97, respectively, and seawater, pack ice, sea-microlayer, subsuperficial water and freshwater, collected during the Italian Expedition of 1997/98. Extractions from the aqueous matrices were carried out in Antarctica (the laboratories of the Italian Base, Terra Nova Bay). Because of the critical space–time conditions in these laboratories, an extraction procedure was developed, suitable for large volumes of water (10 L), in order to combine the extraction of other classes of organic compounds (polychlorinated biphenyls, polycyclic aromatic hydrocarbons and chlorinated pesticides) with those of our direct interest. The VCHC organic extracts were analysed in Italy by GC-ECD and GC-MS. The analyses confirmed the presence of the three halocarbons in Antarctica in quantities ranging from units to some dozens of nanograms per kilogram. The results were evaluated with respect to the local distribution of these compounds and their diffusion on a global scale.

Halocarbons in Antarctic Surface Waters and Snow

Abstract Surface and pit snow samples, collected from Terranova Bay area in Antarctica during the Italian expeditions of 1991–92 and 1992–93, were analysed, for halocarbons, namely tetrachloromethane, trichloroethylene and tetrachloroethylene. The results obtained (including those related with lake and ice water samples previously reported) were evaluated with respect to the worldwide distribution of these compounds and their diffusion to a global scale. Important innovations concerning sensitivity and reproducibility of the analytical method, are also reported.

On-line analysis of volatile chlorinated hydrocarbons in air by gas chromatography–mass spectrometry: Improvements in preconcentration and injection steps

An analytical system composed of a cryofocusing trap injector device coupled to a gas chromatograph with mass spectrometric detection (CTI-GC-MS) specific for the on-line analysis in air of volatile chlorinated hydrocarbons (VCHCs) (dichloromethane; chloroform; 1,1,1-trichloroethane; tetrachloromethane; 1,1,2-trichloroethylene; tetrachloroethylene) was developed. The cryofocusing trap injector was the result of appropriate low cost modifications to an original purge-and-trap device to make it suitable for direct air analysis even in the case of only slightly contaminated air samples, such as those from remote zones. The CTI device can rapidly and easily be rearranged into the purge-and-trap allowing water and air analysis with the same apparatus. Air samples, collected in stainless steel canisters, were introduced directly into the CTI-GC-MS system to realize cryo-concentration (at -120 degrees C), thermal desorption (at 200 degrees C) and for the subsequent analysis of volatiles. The operating phases and conditions were customised and optimized. Recovery efficiency was optimized in terms of moisture removal, cold trap temperature and sampling mass flow. The injection of entrapped volatiles was realized through a direct transfer with high chromatographic reliability (capillary column-capillary column). These improvements allowed obtaining limits of detection (LODs) at least one order of magnitude lower than current LODs for the investigated substances. The method was successfully employed on real samples: air from urban and rural areas and air from remote zones such as Antarctica.

Comparison of atmosphere/aquatic environment concentration ratio of volatile chlorinated hydrocarbons between temperate regions and Antarctica.

For the purpose of understanding the transport and deposition mechanisms and the air-water distribution of some volatile chlorinated hydrocarbons (VCHCs), their atmosphere/aquatic environment concentration ratio was evaluated. In addition, for the purpose of differentiating VCHC behaviour in a temperate climate from its behaviour in a polar climate, the atmosphere/aquatic environment concentration ratio evaluated in matrices from temperate zones was compared with the concentration ratio evaluated in Antarctic matrices. In order to perform air samplings also at rigid Antarctic temperatures, the sampling apparatus, consisting of a diaphragm pump and canisters, was suitably modified. Chloroform, 1,1,1-trichloroethane, tetrachloromethane, 1,1,2-trichloroethylene and tetrachloroethylene were measured in air, water and snow using specific techniques composed of a purpose-made cryofocusing-trap-injector (for air samples) and a modified purge-and-trap injector (for aqueous samples) coupled to a gas chromatograph with mass spectrometric detection operating in selected ion monitoring mode. The VCHCs were retrieved in all the investigated matrices, both Italian and Antarctic, with concentrations varying from tens to thousands of ng m(-3) in air and from digits to hundreds of ng kg(-1) in water and snow. The atmosphere/aquatic environment concentration ratios were always found to be lower than 1. In particular, the Italian air/water concentration ratios were smaller than the Antarctic ones, by reason of the higher atmospheric photochemical activity in temperate zones. On the other hand, the Antarctic air/snow concentration ratios proved to be largely in favour of snow with respect to the Italian ratios, thus corroborating the hypothesis of a more efficient VCHC deposition mechanism and accumulation on Antarctic snow.