Multianalyte LC-MS-based methods in doping control: what are the implications for doping athletes?

Over the last 50 years, the list of doping substances and methods has been progressively expanding, being regularly reviewed by the international antidoping authorities (formerly the Medical Commission of the International Olympic Committee, and afterward, following its constitution in 1999, the World Anti-Doping Agency [WADA]). New substances/classes of substances have been periodically included in the list, keeping the pace with more advanced and sophisticated doping trends. At present, and apart from the prohibited performance enhancing and masking methods (e.g., blood transfusions and tampering strategies), the list comprises several hundreds of biologically active substances, with broad differences in their physicochemical properties (i.e., molecular weight, polarity and acid-basic properties) [1]. As a consequence, the ‘one class – one procedure’ approach, which had been followed by nearly all accredited antidoping laboratories worldwide until the turn of the millennium, is no longer sustainable. The need to minimize the overall number of independent analytical procedures, and, in parallel, to reduce the analytical costs, stimulated the development of multitargeted methods, aimed to increase the overall ratio of ‘target analytes: procedure’ [2–6]. The above evolution has not always been a straight forward process. The need to comply with the WADA technical requirements (both in terms of identification criteria and of minimum required performance limits [7,8]) and with the reduction of the reporting time (a constraint that becomes even more critical during international sport events, where the daily workload also drastically increases) has imposed a thorough re-planning of the analytical procedures. The development of an antidoping analytical method requires the appropriate knowledge not only of the biophysicochemical properties of the target analyte, but also of its PK profile. Historically, immunological methods and GC-based techniques were applied in antidoping science, as preferential screening methods for the detection of prohibited substances, which were originally limited to nonendogenous stimulants and narcotics. In the 1980s, GC–MS became the reference analytical platform for the detection and quantification of the majority of the low molecular weight doping substances [3–6]. In the following two decades, with the inclusion in the Prohibited List of new classes of low molecular weight, hydrophilic, thermolabile, nonvolatile analytes (including, but not limited to, glucocorticoids and designer steroids) and simultaneously of peptide hormones, scientists were obliged to design, develop, validate and apply techniques based on LC–MS/MS

Toxicity testing in environmental monitoring: The role of enzymatic biosensors

Biological toxicity testing is a rapidly expanding field involving numerous bioanalytical techniques. The enzymatic biosensors are valuable screening tools to identify pollutants and/or toxic agents in the environment and/or in food matrices, thus representing a valid alternative to animal testing in analytical toxicology. Inhibition based biosensors here presented have been proved to represent alternative assays for the toxicity evaluation of warfare agents and endocrine disrupting chemicals as well as algal toxins (phycotoxins) in the contamined sea foods (mainly clams and other mollusks). Results obtained by inhibition studies performed by means of several enzymatic biosensors indicate the reliability of the proposed method and the possibility to extend such an experimental approach to other toxicants as a simple, rapid and cheap biotest, to be used easily also "on the spot"

Determination of Endogenous and Synthetic Glucocorticoids in Human Urine by Gas Chromatography – Mass Spectrometry Following Microwave Assisted Derivatization

Acomplete screening and confirmation analytical method for the direct determination of six endogenous (cortisol, cortisone, deoxycorticosterone, tetrahydrocortisol, tetrahydrocortisone, tetrahydro-S) and 17 synthetic (amcinonide, betamethasone, desoximethasone, dexamethasone, fludrocortisone, flumethasone, flunisolide, flucinolone acetonide, flucinonide, fluprednisolone, flurandrenolide, fluorometholone, 6-methylprednisolone, prednisolone, prednisone, triamcinolone, triamcinolone acetonide) glucocorticoids in human urine by gas chromatography with mass spectrometric detection (GC–MS) is presented. The analytical technique comprises a pre treatment procedure and the instrumental analysis of the trimethylsilyl (TMS) derivatives, performed by GC–MS (quadrupole) with electron impact (EI) ionization. The derivatization yields obtained by two different derivatizing mixtures, namely N-methyl N(trimethylsilyl)trifluoroacetamide (MTSFA):NH4I:dithioerythritol (DTE) 1000:2:4 (usually indicated as TMSiodine); and N-trimethylsilylimidazole (TMSim):N,O-bis(trimethylsilyl)acetamide (BSA):trimethylchlorosilane (TMCS) 3:3:2, both under direct thermal heating and with microwave (MW) irradiation, were evaluated, also as a function of the temperature, of the MWì power and of the incubation time. The highest yields of the derivatization process were obtained, for most of the compounds here considered, by a two-step procedure: a microwave-assisted derivatization stage (40 min in a microwave oven at 900Wemitted power), followed by a traditional heat transfer derivatization (1.5 h in a thermostated bath at 70 ◦C) with the derivatization mixture TMSim:BSA:TMCS 3:3:2. In these operating conditions, diagnostic EI–MS spectra of all considered glucocorticoids were obtained. Limits of detection (LOD) of synthetic glucocorticoids in urine ranged from 3 to 25g/l. The effectiveness of the method for the determination of glucocorticoids in urine was evaluated on spiked urine samples and on real samples obtained from patients under pharmacological treatment with synthetic glucocorticoids. Apart from the clinical monitoring of glucocorticoids in urine, the method can be applied as a complete screening + confirmation analytical protocol in antidoping tests for the detection of illicit administration of glucocorticoids by the athletes

Ecdysteroids: A novel class of anabolic agents?

Increasing numbers of dietary supplements with ecdysteroids are marketed as “natural anabolic agents”. Results of recent studies suggested that their anabolic effect is mediated by estrogen receptor (ER) binding. Within this study the anabolic potency of ecdysterone was compared to well characterized anabolic substances. Effects on the fiber sizes of the soleus muscle in rats as well the diameter of C2C12 derived myotubes were used as biological readouts. Ecdysterone exhibited a strong hypertrophic effect on the fiber size of rat soleus muscle that was found even stronger compared to the test compounds metandienone (dianabol), estradienedione (trenbolox), and SARM S 1, all administered in the same dose (5 mg/kg body weight, for 21 days). In C2C12 myotubes ecdysterone (1 µM) induced a significant increase of the diameter comparable to dihydrotestosterone (1 µM) and IGF 1 (1.3 nM). Molecular docking experiments supported the ERβ mediated action of ecdysterone. To clarify its status in sports, ecdysterone should be considered to be included in the class “S1.2 Other Anabolic Agents” of the list of prohibited substances of the World Anti-Doping Agency

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-MS3 (ion trap) with electron impact ionization. The GC-MS3 analysis allows isolation and characterization of specific fragments from the original (MS1) molecular structure, and in particular, those fragments originating from the precursor ion cluster (m/z = 335-337) characteristic of clenbuterol. The MS2 product fragment m/z = 300 is in turn used as a further precursor fragment giving rise to a MS3 spectrum specific for clenbuterol. MS4 fragmentation spectra were also investigated. However, further fragmentation of MS3 product ions does not lead to functional MS4 spectra nor to any significant increase in the signal-to-noise ratio. The sensitivity limit of the MS3 technique is lower than 0.2 mug/l, with a linear range between 0.5 and 5 mug/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. (C) 2002 Elsevier Science B.V. All rights reserved

Rapid Determination of Diuretics in Human Urine by Gas Chromatography – Mass Spectrometry Following Microwave Assisted Derivatization

This work presents a GC–MS–MS–MS method for the direct determination of clenbuterol in human urine. The method 3 comprises a pretreatment procedure and the instrumental analysis of the derivatives performed by GC–MS (ion trap) with 3 electron impact ionization. The GC–MS analysis allows isolation and characterization of specific fragments from the 1 original (MS ) molecular structure, and in particular, those fragments originating from the precursor ion cluster (m/z5335– 2 337) characteristic of clenbuterol. The MS product fragment m/z5300 is in turn used as a further precursor fragment 3 4 giving rise to a MS spectrum specific for clenbuterol. MS fragmentation spectra were also investigated. However, further 3 4 fragmentation of MS product ions does not lead to functional MS spectra nor to any significant increase in the 3 signal-to-noise ratio. The sensitivity limit of the MS technique is lower than 0.2 mg/ l, with a linear range between 0.5 and 5 mg/ 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

Rapid screening of beta-adrenergic agents and related compounds in human urine for anti-doping purpose using capillary electrophoresis with dynamic coating

This paper presents a capillary electrophoresis method, developed for the detection, in human urine, of beta-adrenergic agents and phenolalkylamines. The electrophoretic separation is achieved in less than 10 min and is based on the use of CEofix kit, for the dynamic capillary coating. The effects of accelerator buffer pH and separation voltage were investigated. The optimum buffer pH was found to be 2.5 for beta2-agonists and 6.2 for beta-blockers and phenoalkylamines with a separation voltage of 15 IN. Urine samples spiked with the compounds here studied were treated according to the standard procedure (SPE and evaporation to dryness) and analyzed by CE interfaced with an UV diode-array, set at 195 and 210 nm. The quantitative validation results, obtained analyzing samples at three different concentrations, show a good precision of peak areas that do not exceed 5% for intra-day assays and 10% for inter-day assays. Good linearity (r(2) > 0.995) was obtained within the 50-500 ng/mL concentration range. The qualitative validation data show a relative migration times (MTs) variation lower than 1%. The analytes were clearly distinguishable in urine, with LOD and LOQ in the range of 10-80 and 40-100 ng/mL, respectively

The use of cytochrome P450 inhibitors in sport. A new generation of doping masking agents?

The activity of the CYP450 enzymes responsible for the phase I metabolism of most of the compounds included in the World Anti-Doping Agency (WADA) list of prohibited substances and methods could be strongly modified by the combined administration of other drugs such as, for example, the antidepressant, the antifungal and the H2 receptor antagonist agents. These compounds act as inhibitors of the CYP450 isoforms and it has been demonstrated that their co-administration with a drug that is also a CYP450 substrate may lead to a substantial alteration of the latter drug bioavailability, metabolism and excretion kinetics. In sports some classes of non-banned drugs, and primarily among them antidepressants, antifungals and the H2 receptor antagonists are extensively used, according to the information available on the doping control forms. Athletes may intentionally combine the CYP450 inhibitors with doping agents to modify in urine the time window of detection of the selected marker(s) of drug abuse, especially in those cases where the parent drugs are extensively metabolized

Drug Use on Mont Blanc: A Study Using Automated Urine Collection

Mont Blanc, the summit of Western Europe, is a popular but demanding high-altitude ascent. Drug use is thought to be widespread among climbers attempting this summit, not only to prevent altitude illnesses, but also to boost physical and/or psychological capacities. This practice may be unsafe in this remote alpine environment. However, robust data on medication during the ascent of Mont Blanc are lacking. Individual urine samples from male climbers using urinals in mountain refuges on access routes to Mont Blanc (Goûter and Cosmiques mountain huts) were blindly and anonymously collected using a hidden automatic sampler. Urine samples were screened for a wide range of drugs, including diuretics, glucocorticoids, stimulants, hypnotics and phosphodiesterase 5 (PDE-5) inhibitors. Out of 430 samples analyzed from both huts, 35.8% contained at least one drug. Diuretics (22.7%) and hypnotics (12.9%) were the most frequently detected drugs, while glucocorticoids (3.5%) and stimulants (3.1%) were less commonly detected. None of the samples contained PDE-5 inhibitors. Two substances were predominant: the diuretic acetazolamide (20.6%) and the hypnotic zolpidem (8.4%). Thirty three samples were found positive for at least two substances, the most frequent combination being acetazolamide and a hypnotic (2.1%). Based on a novel sampling technique, we demonstrate that about one third of the urine samples collected from a random sample of male climbers contained one or several drugs, suggesting frequent drug use amongst climbers ascending Mont Blanc. Our data suggest that medication primarily aims at mitigating the symptoms of altitude illnesses, rather than enhancing performance. In this hazardous environment, the relatively high prevalence of hypnotics must be highlighted, since these molecules may alter vigilance