Search

The urinary steroidal fraction has been extensively explored as non-invasive alternative to monitor pathological conditions as well as to unveil the illicit intake of pseudo-endogenous anabolic steroids in sport. However, the majority of previous approaches involved the a priori selection of potentially relevant target analytes. Here we describe the non-targeted analysis of the urinary steroidal profiles. The workflow includes minimal sample pretreatment and normalization according to the specific gravity of urine, a 20 min reverse phase ultra-performance liquid chromatographic separation hyphenated to electrospray time-of-flight mass spectrometry. As initial validation, we analyzed a set of quality control urines spiked with glucurono- and sulfo-conjugated steroids at physiological ranges. We then applied the method for the analysis of samples collected after single transdermal administration of testosterone in hypogonadal men. The method allowed profiling of approximately three thousand metabolic features, including steroids of clinical and forensic relevance. It successfully identified metabolic pathways mostly responsible for groups clustering even in the context of high inter-individual variability and allowed the detection of currently unknown metabolic features correlating with testosterone administration. These outcomes set the stage for future studies aimed at implementing currently monitored urinary steroidal markers both in clinical and forensic analysis.

Stimulants are banned by the World Anti-Doping Agency (WADA) if used “in competition”. Being the analysis of stimulants presently carried out on urine samples only, it might be useful, for a better interpretation of analytical data, to discriminate between an early intake of the substance and an administration specifically aimed to improve the sport performance. The purpose of the study was to investigate the differences, in terms of excretion/disappearance of drugs, between urine and oral fluid, a sample that can reflect plasmatic concentrations. Oral fluid and urine samples were collected following oral administration of the following stimulants: modafinil (100 mg), selegiline (10 mg), crotetamide/cropropamide (50 mg each), pentetrazol (100 mg), ephedrine (12 mg), sibutramine (10 mg), mate de coca (a dose containing about 3 mg of cocaine); analysis of drugs/metabolites was carried out by gas chromatography/mass spectrometry (GC/MS) in both body fluids. Our results show that both the absolute concentrations and their variation as a function of time, in urine and in oral fluid, are generally markedly different, being the drugs eliminated from urine much more slowly than from oral fluid. Our results also suggest that the analysis of oral fluid could be used to successfully complement the data obtained from urine for “in competition” anti-doping tests; in all those cases in which the metabolite(s) concentration of a substance in urine is very low and the parent compound is not detected, it is indeed impossible, relying on urinary data only, to discriminate between recent administrations of small doses and remote administrations of higher doses.

This work presents a complete method for the screening and confirmation analysis of diuretics in human urine by gas chromatography–mass spectrometry (GC–MS). The method comprises a pretreatment stage (extraction, preconcentration and derivatization to form the corresponding methyl derivatives) and the subsequent analysis of the derivatized extracts by GC–MS. Particularly, the derivatization stage, necessary to form the methyl derivatives of the compounds detectable by the GC–MS technique, is carried out under microwave irradiation rather than with direct thermal heating, thus reducing the incubation time from 3 h to 10 min. Microwave assisted derivatization also allowed an improvement of the limits of detection (LODs) for all the compounds here considered. The technique is particularly suitable for the rapid analysis of huge population of samples, as it is the case of urine analysis by the antidoping laboratories, and especially in all those occasions where rapid response times are requested.

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.

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.

This work presents the realization of enzymatic bioelectrodes, suitable for the determination of γ-aminobutyric acid (GABA). The biosensors are based on the catalytic activity of the enzymes γ-aminobutyric glutamic transaminase (GABA-T), succinic semialdehyde dehydrogenase (SSDH) and horseradish peroxidase (HPO). The first two enzymes are usually indicated by the general term “GABASE”. All the biosensors presented in this work are realized by immobilizing the enzyme HPO on the tip of an amperometric oxygen selective electrode: the resulting NADPH-sensitive biosensor is used in combination with GABASE to determine the concentration of GABA in aqueous samples. Since SSDH depends on the NADP+/NADPH equilibrium, it follows that, in the presence of HPO, the NADPH formed is oxidized to NADP, and the decrease in the concentration of dissolved oxygen is proportional to the concentration of NADPH and, in turn, to that of GABA. The experiments were performed either with GABASE free in solution or co-immobilized with HPO on the surface of the oxygen electrode. In the latter case, the immobilization of the three enzymes has been performed either on a single membrane or on two separated membranes. In both cases there is an almost perfect linearity between the electrode signal and GABA concentration in the range 5.0 × 10−5–1.2 × 10−3M, with a lower detection limit of 2.0 × 10−5M; but the single-membrane biosensor showed a better overall performance, especially in terms of repeatability of measurements and lifetime of operation.

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.