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This paper presents a general screening method, based on liquid chromatography/mass spectrometry (LC/MS), for the simultaneous detection in human urine of 72 xenobiotics (21 diuretics, 16 synthetic glucocorticoids, 17 beta-adrenergic drugs, 10 stimulants, 5 anti-oestrogens and 3 anabolic steroids), excreted free or as glucuro-conjugates in urine. Although the method has been specifically designed and evaluated in view of its potential application to anti-doping analyses, it can also be effective in other areas of analytical toxicology. Sample preparation was based on two liquid/liquid separation steps (performed at alkaline and at acid pH, respectively) of hydrolyzed human urine, and then an assay by LC/MS-MS in positive and negative ionization mode using an electrospray ionization source (ESI) and multiple reaction monitoring (MRM) as the acquisition mode. The overall time needed for an LC run was less than 15 minutes. All compounds showed good reproducibility in terms of both the retention times (CV%<1) and the relative abundances of the diagnostic transitions (CV%<10). The limits of detection (LOD) were in the range of 1-50 ng/mL for glucocorticoids, anti-oestrogens and steroids, and 50-500 ng/mL for diuretics, beta-adrenergic drugs and stimulants, thus satisfying the minimum required performance limits (MRPL) set by the World Anti-Doping Agency (WADA) for the accredited anti-doping laboratories.

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 Agency's (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.

A method for the toxicological screening of the new designer drug methylenedioxypyrovalerone (MDPV) is described; with an emphasis on its application for anti-doping analysis. The metabolism of MDPV was evaluated in vitro using human liver microsomes and S9 cellular fractions for CYP450 phase I and uridine 5'-diphosphoglucuronosyltransferase (UGT) and sulfotransferase (SULT) phase II metabolism studies. The resulting metabolites were subsequently liquid/liquid extracted and analyzed using gas chromatography/mass spectrometry (GC/MS) as trimethylsilyl (TMS) derivatives. The structures of the metabolites were further confirmed by accurate mass measurement using a liquid chromatography/quadrupole time-of-flight (LC/QTOF) mass spectrometer. The studies demonstrated that the main metabolites of MDPV are catechol and methyl catechol pyrovalerone, which are in turn sulfated and glucuronated. The method for the determination of MDPV in urine has been fully validated by assessing the limits of detection and quantification, linearity, repeatability, and accuracy. This validation demonstrates the suitability for screening of this stimulant substance for anti-doping and forensic toxicology purposes.

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Recent studies suggest that the anabolic effect of ecdysterone, a naturally occurring steroid hormone claimed to enhance physical performance, is mediated by estrogen receptor (ER) binding. In comparison with the prohibited anabolic agents (e.g., metandienone and others), ecdysterone revealed to be even more effective in a recent study performed in rats. However, scientific studies in humans are very rarely accessible. Thus, our project aimed at investigating the effects of ecdysteronecontaining products on human sport exercise. A 10-week intervention study of strength training of young men (n = 46) was carried out. Different doses of ecdysterone-containing supplements have been administered during the study to evaluate the performance-enhancing effect. Analysis of blood and urine samples for ecdysterone and potential biomarkers of performance enhancement has been conducted. To ensure the specificity of the effects measured, a comprehensive screening for prohibited performance-enhancing substances was also carried out. Furthermore, the administered supplement has been tested for the absence of anabolic steroid contaminations prior to administration. Significantly higher increases in muscle mass were observed in those participants that were dosed with ecdysterone. The same hypertrophic effects were also detected in vitro in C2C12 myotubes. Even more relevant with respect to sports performance, significantly more pronounced increases in one-repetition bench press performance were observed. No increase in biomarkers for liver or kidney toxicity was noticed. These data underline the effectivity of an ecdysterone supplementation with respect to sports performance. Our results strongly suggest the inclusion of ecdysterone in the list of prohibited substances and methods in sports in class S1.2 "other anabolic agents".

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In this paper, we discuss the capabilities of liquid chromatography coupled to mass spectrometry with a time-of flight system with accurate mass measurement for the detection and characterisation of drug metabolites in biological samples for anti-doping purpose. Urinary excretion samples of three selective oestrogen receptor modulators (SERMs) with a common triphenylethylene structure: clomiphene, toremifene, and tamoxifen, obtained after oral administration of a single dose of each drug, were analysed using a time-of-flight system, after automatic tuning and calibration of the equipment, in positive full scan mode using an electrospray ionisation source. Following this approach we detected most of all significant metabolites reported by others and postulated new metabolites, especially for toremifene, have been characterised: N-demethyl-3-hydroxy-4-methoxy-toremifene and 3- hydroxy-4-methoxy-toremifene; in addtiona to this, in the urinary excretion samples of toremifene some metabolites, without the characteristic chlorine isotope pattern, discarded in previous studies, that are also metabolites of tamoxifen, were identified. The lack of certified reference materials does not allow an accurate determination of the limit of detection (LODs) of all metabolites; however an estimation taking into account the response factor of similar compounds allows to estimate that all metabolites are clearly detectable in a range of concentration comprised between 10 ng mL(-1) and 30 ng mL(-1).