No abstract

The discovery and implementation of the long-term metabolite of metandienone, namely 17β-hydroxymethyl-17α-methyl-18-norandrost-1,4,13-trien-3-one, to doping control resulted in hundreds of positive metandienone findings worldwide and impressively demonstrated that prolonged detection periods significantly increase the effectiveness of sports drug testing. For oxandrolone and other 17-methyl steroids, analogs of this metabolite have already been described, but comprehensive characterization and pharmacokinetic data are still missing. In this report, the synthesis of the two epimeric oxandrolone metabolites-17β-hydroxymethyl-17α-methyl-18-nor-2-oxa-5α-androsta-13-en-3-one and 17α-hydroxymethyl-17β-methyl-18-nor-2-oxa-5α-androsta-13-en-3-one-using a fungus (Cunninghamella elegans) based protocol is presented. The reference material was fully characterized by liquid chromatography nuclear magnetic resonance spectroscopy and high resolution/high accuracy mass spectrometry. To ensure a specific and sensitive detection in athlete's urine, different analytical approaches were followed, such as liquid chromatography-tandem mass spectrometry (QqQ and Q-Orbitrap) and gas chromatography-tandem mass spectrometry, in order to detect and identify the new target analytes. The applied methods have demonstrated good specificity and no significant matrix interferences. Linearity (R(2) > 0.99) was tested, and precise results were obtained for the detection of the analytes (coefficient of variation <20%). Limits of detection (S/N) for confirmatory and screening analysis were estimated at 1 and 2 ng/mL of urine, respectively. The assay was applied to oxandrolone post-administration samples to obtain data on the excretion of the different oxandrolone metabolites. The studied specimens demonstrated significantly longer detection periods (up to 18 days) for the new oxandrolone metabolites compared to commonly targeted metabolites such as epioxandrolone or 18-nor-oxandrolone, presenting a promising approach to improve the fight against doping.

Increasing the blood's capacity for oxygen transport by erythropoiesis‐stimulating agents (ESAs) constitutes a prohibited procedure of performance enhancement according to the World Anti‐Doping Agency (WADA). The advent of orally bio‐available small‐molecule ESAs such as hypoxia‐inducible factor (HIF) stabilizers in the development of novel anti‐anaemia therapies expands the list of potential ESA doping techniques. Here, the erythropoiesis‐stimulating properties and doping relevance of experimental HIF‐stabilizers, such as cobaltous chloride, 3,4‐dihydroxybenzoic acid or GSK360A, amongst others, are discussed. The stage of clinical trials is reviewed for the anti‐anaemia drug candidates FG‐2216, FG‐4592, GSK1278863, AKB‐6548, and BAY85‐3934. Currently available methods and strategies for the determination of selected HIF stabilizers in sports drug testing are based on liquid chromatography‐electrospray ionization‐tandem mass spectrometry (LC‐ESI‐MS/MS). For the support of further analytical assay development, patents claiming distinct compounds for the use in HIF‐mediated therapies are evaluated and exemplary molecular structures of HIF stabilizers presented. Moreover, data concerning the erythropoiesis‐enhancing effects of the GATA inhibitors K7174 and K11706 as well as the lipidic small‐molecule ESA PBI‐1402 are elucidated the context of doping analysis. Copyright © 2012 John Wiley & Sons, Ltd.

The study of the collision-induced dissociation behavior of various substituted isoquinoline-3-carboxamides, which are amongst a group of drug candidates for the treatment of anemic disorders (e.g., FG-2216), allowed for the formulation of the general mechanisms underlying the unusual fragmentation behavior of this class of compounds. Characterization was achieved with high-resolution/high accuracy LTQ-Orbitrap tandem mass spectrometry of the protonated precursor ions. Presented data were substantiated by the synthesis and analysis of proposed gas-phase intermediate structures and stable isotope labeled analogues, as well as by density functional theory calculations. Exemplary, CID of protonated N-[(1-chloro-4-hydroxy-7-isopropoxy-isoquinolin-3-yl)carbonyl]glycine gives rise to the isoquinoline-3-carboxy-methyleneamide product ion which nominally eliminates a fragment of 11 u. This was attributed to the loss of methyleneamine (Ϫ29 u) and a concomitant spontaneous and reversible water addition (ϩ18 u) to the resulting acylium ion to yield the protonated isoquinoline-3-carboxylic acid. The same water addition pattern is observed after loss of propylene (Ϫ42 u). A further nominal loss of 10 u is explained by the elimination of carbon monoxide (Ϫ28 u) followed by another water adduct formation (ϩ18 u) to yield the protonated 1-chloro-3,4,7-trihydroxy-isoquinoline. The source of the multiple gas-phase water addition remained unclear. This atypical fragmentation pattern proved to be highly characteristic for all studied and differentially substituted isoquinoline-3-carboxamides, and offers powerful analytical tools for the establishment of a LC/MS(/MS) based screening procedure for model HIF-stabilizers and their potential metabolites in clinical, forensic and sports drug testing. (J Am Soc Mass

Selective androgen receptor modulators (SARMs) are potent anabolic agents with tissue-selective properties. Due to their potential misuse in elite sport, the World Anti-Doping Agency (WADA) has prohibited SARMs since 2008, and although no representative drug candidate has yet received full clinical approval, recent findings of SARMs illegally sold via the internet have further supported the need to efficiently test for these compounds in doping controls. In the present communication, the mass spectrometric characterization of urinary metabolites of the SARM Andarine (also referred to as S-4) compared with earlier in vitro and animal studies is reported. Liquid chromatography interfaced to high-resolution/high-accuracy (tandem) mass spectrometry was used to identify phase I and II metabolites, confirming the predicted target analytes for sports drug testing purposes including the glucuronic acid conjugates of the active drug, its monohydroxylated and/or deacetylated product, the hydrolysis product resulting from the removal of the compound's B-ring, as well as the sulfate of the monohydroxylated and the deacetylated phase I metabolite. The obtained data will support future efforts to effectively screen for and confirm the misuse of the non-approved drug candidate Andarine.