Sports Drug Testing Research Articles

Recent developments in the use of isotope ratio mass spectrometry in sports drug testing

According to the annual report of the World Anti-Doping Agency, steroids are the most frequently detected class of doping agents. Detecting the misuse of endogenously occurring steroids, i.e. steroids such as testosterone that are produced naturally by humans, is one of the most challenging issues in doping control analysis. The established thresholds for urinary concentrations or concentration ratios such as the testosterone/epitestosterone quotient are sometimes inconclusive owing to the large biological variation in these parameters.For more than 15 years, doping control laboratories focused on the carbon isotope ratios of endogenous steroids to distinguish between naturally elevated steroid profile parameters and illicit administration of steroids. A variety of different methods has been developed throughout the last decade and the number of different steroids under investigation by isotope ratio mass spectrometry has recently grown considerably. Besides norandrosterone, boldenone was found to occur endogenously in rare cases and the misuse of corticosteroids or epitestosterone can now be detected with the aid of carbon isotope ratios as well. In addition, steroids excreted as sulfoconjugates were investigated, and the first results regarding hydrogen isotope ratios recently became available.All of these will be presented in detail within this review together with some considerations on validation issues and on identification of parameters influencing steroidal isotope ratios in urine.

Response to Letter to the Editor regarding “Rapid determination of urinary di(2-ethylhexyl) phthalate metabolites based on liquid chromatography/tandem mass spectrometry as a marker for blood transfusion in sports drug testing”

Response to Letter to the Editor regarding “Rapid determination of urinary di(2-ethylhexyl) phthalate metabolites based on liquid chromatography/tandem mass spectrometry as a marker for blood transfusion in sports drug testing”

Letter to the Editor regarding “Rapid determination of urinary di(2-ethylhexyl) phthalate metabolites based on liquid chromatography/tandem mass spectrometry as a marker for blood transfusion in sports drug testing”

Letter to the Editor regarding “Rapid determination of urinary di(2-ethylhexyl) phthalate metabolites based on liquid chromatography/tandem mass spectrometry as a marker for blood transfusion in sports drug testing”

Current role of LC-MS(/MS) in doping control

Liquid chromatography-(tandem) mass spectrometry [(LC-MS(/MS)] has become an integral part of modern sports drug testing as it offers unique capabilities complementing immunological and gas chromatography-(tandem) mass spectrometry [(GC-MS(/MS)]-based detection methods for prohibited compounds. The improved options of fast and sensitive targeted analysis as well as untargeted screening procedures utilizing high resolution/high accuracy mass spectrometry have considerably expanded the tools available to anti-doping laboratories for initial testing and confirmation methods. One approach is to focus on pre-selected target analytes that are measured with utmost specificity and sensitivity using diagnostic precursor-product ion pairs in low resolution tandem mass spectrometers. The other scenario is to measure and plot extracted ion chromatograms of protonated or deprotonated molecules as well as product ions as recorded in the full scan mode with high resolution/high accuracy mass spectrometry. Examples of recent applications of sports drug testing procedures published between 2007 and 2010 are presented and discussed, outlining the particular advantages of the selected approaches as well as their limitations in a short- and long-term perspective.

Developing an Immunoassay for the Anabolic Steroid Stanozolol

As part of the UK sports drug testing programme, urine collected from competitors is screened at the Drug Control Centre for the presence of anabolic steroids by gas chromatography/mass spectrometry (GC/MS). Stanozolol (17α-methyl-pyrazo-(4′,5′;2,3)-5-androstan-17β-ol) is an anabolic steroid conferred oral activity by the 17α-methyl substituent. Stanozolol and its metabolites are difficult to detect reliably by GC/MS without careful optimization of the procedure. Hence an immunoassay for the detection of stanozolol is being developed as an alternative. This paper describes the strategy behind immunogen formation (prior to immunization) and preparation of the radio-label.

Experimental and Kinetic Studies of the Escherichia coli Glucuronylsynthase: An Engineered Enzyme for the Synthesis of Glucuronide Conjugates

The detection and study of glucuronide metabolites is essential in many fields including pharmaceutical development, sports drug testing, and the detection of agricultural residues. Therefore, the development of improved methods for the synthesis of glucuronide conjugates is an important aim. The glycosynthase derived from E. coli β-glucuronidase provides an efficient, scalable, single-step synthesis of β-glucuronides under mild conditions. In this article we report on experimental and kinetic studies of the E. coli glucuronylsynthase, including the influence of acceptor substrate, pH, temperature, cosolvents, and detergents, leading to optimized conditions for glucuronide synthesis. Enzyme kinetics also reveals that both substrate and product inhibition may occur in glucuronylsynthase reactions but that these effects can be ameliorated through the judicious choice of acceptor and donor substrate concentrations. An investigation of temporary polar substituents was conducted leading to improved aqueous solubility of hydrophobic steroidal acceptors. In this way the synthesis of the steroidal metabolite dehydroepiandrosterone 3-β-D-glucuronide was achieved in three steps and 86% overall yield from dehydroepiandrosterone.

Mario Thevis: Mass spectrometry in sports drug testing. Characterization of prohibited substances and doping control analytical assays

Mario Thevis: Mass spectrometry in sports drug testing. Characterization of prohibited substances and doping control analytical assays

Determination of IGF-1 and IGF-2, their degradation products and synthetic analogues in urine by LC-MS/MS

Peptide analysis in doping controls by means of nano-UPLC coupled high resolution/high mass accuracy mass spectrometry provides the state-of-the-art technique in modern sports drug testing. The present study describes a recent application of this technique for the qualitative determination of different urinary insulin-like growth factor (IGF) related peptides. After simultaneous isolation by solid phase extraction and magnetic particle-based immunoaffinity purification, target analytes (IGF-1, IGF-2, Des1-3-IGF-1, R(3)-IGF-1 and longR(3)-IGF-1) were separated by nano-liquid chromatography prior to mass spectrometric detection. Endogenously produced IGF-1 and IGF-2, as well as the degradation product Des1-3-IGF-1, were frequently detected in urine samples from healthy volunteers in a concentration range of 20-400 pg mL(-1). The impact of IGF binding proteins (IGFBPs), being also present in urine, was potentially estimated by an additional ultrafiltration step in the sample preparation procedure. The synthetic analogue longR(3)-IGF-1, which is assumed to be subject to misuse by cheating athletes, was also analysed and detected in fortified urine samples. Besides the intact molecule, an N-terminally truncated degradation product Des1-10-longR(3)-IGF-1 was identified as the more stable target for doping controls using urine samples. The method was validated for qualitative purposes considering the parameters specificity, limit of detection (20-50 pg mL(-1)), recovery (10-35%), precision (<20%), linearity, robustness and stability.

Annual banned‐substance review: analytical approaches in human sports drug testing

The timely update of the list of prohibited substances and methods of doping (as issued by the World Anti-Doping Agency) is an essential aspect of international anti-doping efforts and represents consensual agreement by expert panels regarding substances and the methods of performance manipulation in sports. The annual banned-substance review for human doping controls critically summarizes recent innovations in analytical approaches; its purpose is to improve the quality of doping controls by reporting emerging and advancing methods that focus on detecting known and recently outlawed substances. This review surveys new and/or enhanced procedures and techniques of doping analysis together with information relevant to doping control that has been published in the literature between October 2009 and September 2010.

The detection of doping by means of chromatographic methods

AbstractThis article was first published in German in Der Sportarzt, 1966, 2, 81–84 as Der Dopingnachweis mit Hilfe chromatographischer Methoden. Translated and republished with permission.Doping has become an issue in elite sport and necessitates sensitive detection assays that enable the identification of organic compounds on a microscale level in urine. In agreement with modern toxicological methods, sports drug testing approaches can utilize paper, thin layer or gas chromatographic methods to reveal the presence of prohibited substances such as strychnine, pervitine, captagone, benzedrine etc. in doping control specimens. Basic principles of these strategies are summarized and considerations for future applications discussed. Copyright © 2010 John Wiley &amp; Sons, Ltd.

Rapid determination of urinary di(2-ethylhexyl) phthalate metabolites based on liquid chromatography/tandem mass spectrometry as a marker for blood transfusion in sports drug testing

Methods of blood doping such as autologous and homologous blood transfusion are one of the main challenging doping practices in competitive sport. Whereas homologous blood transfusion is detectable via minor blood antigens, the detection of autologous blood transfusion is still not feasible. A promising approach to indicate homologous or autologous blood transfusion is the quantification of increased urinary levels of di(2-ethylhexyl) phthalate (DEHP) metabolites found after blood transfusion. The commonly used plasticizer for flexible PVC products, such as blood bags, is DEHP which is known to diffuse into the stored blood. Therefore, a straight forward, rapid and reliable assay is presented for the quantification of the main metabolites mono(2-ethyl-5-oxohexyl) phthalate, mono(2-ethyl-5-hydroxyhexyl) phthalate and mono(2-ethylhexyl) phthalate that can easily be implemented into existing multi-target methods used for sports drug testing. Quantification of the DEHP metabolites was accomplished after enzymatic hydrolysis of urinary glucuronide conjugates and direct injection using isotope-dilution liquid chromatography/tandem mass spectrometry. The method was fully validated for quantitative purposes considering the parameters specificity, linearity (1-250 ng/mL), inter- (2.4%-4.3%) and intra-day precision (0.7%-6.1%), accuracy (85%-105%), limit of detection (0.2-0.3 ng/mL), limit of quantification (1 ng/mL), stability and ion suppression effects. Urinary DEHP metabolites were measured in a control group without special exposure to DEHP (n = 100), in hospitalized patients receiving blood transfusion (n = 10), and in athletes (n = 468) being subject of routine doping controls. The investigation demonstrates that significantly increased levels of secondary DEHP metabolites were found in urine samples of transfused patients, strongly indicating blood transfusion.

Screening for benfluorex and its major urinary metabolites in routine doping controls

Benfluorex [1-(m-trifluoromethylphenyl)-2-(β-benzoyloxyethyl)aminopropane] has been widely used for the treatment of atherogenic metabolic disorders and impaired carbohydrate metabolism (particularly in obese type-II diabetic patients) as well as an anorectic drug. Due to its potentially performance-enhancing properties, benfluorex has been added to the list of prohibited compounds and methods of doping by the World Anti-Doping Agency (WADA) in 2010, necessitating the implementation of the drug as well as its major metabolites into routine doping control procedures. In the present study, human urinary metabolites of benfluorex were characterized by gas chromatography-electron ionization-mass spectrometry (GC-EI-MS) as well as liquid chromatography-electrospray ionization-high resolution/high accuracy tandem mass spectrometry (LC-ESI-MS/MS). Commonly employed sports drug testing approaches consisting of liquid-liquid extraction followed by GC-MS or urine dilution and immediate LC-MS/MS analysis were expanded and validated with regard to specificity, recovery (48-54%, GC-MS only), intra- and interday precision (<25%), limits of detection (5-8 ng/mL for LC-MS/MS and 80 ng/mL for GC-MS), and ion suppression (for LC-ESI-MS/MS only) to allow the detection of benfluorex metabolites 1-(m-trifluoromethylphenyl)-2-(2-hydroxyethyl)aminopropane (M1), 1-(m-trifluoromethylphenyl)-2-(2-carboxymethyl)aminopropane (M2), and 1-(m-trifluoromethylphenyl)-2-aminopropane (M3) as well as the glucuronic acid conjugate of M1.

Advances in sports drug testing: An overview

Advances in sports drug testing: An overview

Characterization of in vitro generated metabolites of the selective androgen receptor modulators S‐22 and S‐23 and in vivo comparison to post‐administration canine urine specimens

Selective androgen receptor modulators (SARMs) have great therapeutic potential in various diseases including cancer cachexia, sarcopenia, and osteoporosis, and the number of drug candidates has been growing over the last decade. The SARM drug candidates S-22 and S-23 belong to one of the most advanced groups of androgen receptor modulators and are based on an arylpropionamide-derived core structure. Due to their anabolic effects, SARMs have been prohibited in elite sports and have been a subject of sports drug testing programmes since January 2008. Consequently, the structure of analytically useful urinary metabolites should be elucidated to provide targets for sensitive and retrospective analysis. In the present study, the phase-I and -II metabolism of S-22 and S-23 was simulated using hepatic human enzymes, and resulting metabolites were characterized by means of state-of-the-art mass spectrometric approaches employing high resolution/high accuracy Orbitrap mass spectrometry. Subsequently, the newly defined target compounds including the glucuronic acid conjugates of S-22 and S-23, their corresponding monohydroxylated and bishydroxylated analogs, as well as their B-ring depleted counterparts were implemented into an existing routine doping control procedure, which was examined for its specificity for the added substances. In order to obtain proof-of-concept data for authentic urine specimens, canine urine samples collected up to 72 h after oral administration of S-22 to dogs were analyzed using the established approach outlining the capability of the presented assay to detect the glucuronide of S-22 as well as the B-ring-depleted metabolite (M3) in all samples following therapeutic (31.4 µg/kg) dosing. Finally, M3 was chemically synthesized, characterized by nuclear magnetic resonance spectroscopy and high resolution/high accuracy mass spectrometry, and chosen as primary target for future doping control analyses.

Screening for the synthetic cannabinoid JWH‐018 and its major metabolites in human doping controls

Referred to as 'spice', several new drugs, advertised as herbal blends, have appeared on the market in the last few years, in which the synthetic cannabinoids JWH-018 and a C(8) homologue of CP 47,497 were identified as major active ingredients. Due to their reported cannabis-like effects, many European countries have banned these substances. The World Anti-Doping Agency has also explicitly prohibited synthetic cannabinoids in elite sport in-competition. Since urine specimens have been the preferred doping control samples, the elucidation of the metabolic pathways of these substances is of particular importance to implement them in sports drug testing programmes. In a recent report, an in vitro phase-I metabolism study of JWH-018 was presented yielding mainly hydroxylated and N-dealkylated metabolites. Due to these findings, a urine sample of a healthy man declaring to have smoked a 'spice' product was screened for potential phase-I and -II metabolites by high-resolution/high-accuracy mass spectrometry in the present report. The majority of the phase-I metabolites observed in earlier in vitro studies of JWH-018 were detected in this urine specimen and furthermore most of their respective monoglucuronides. As no intact JWH-018 was detectable, the monohydroxylated metabolite being the most abundant one was chosen as a target analyte for sports drug testing purposes; a detection method was subsequently developed and validated in accordance to conventional screening protocols based on enzymatic hydrolysis, liquid-liquid extraction, and liquid chromatography/electrospray tandem mass spectrometry analysis. The method was applied to approximately 7500 urine doping control samples yielding two JWH-018 findings and demonstrated its capability for a sensitive and selective identification of JWH-018 and its metabolites in human urine.

Mass spectrometric characterization of urinary metabolites of the selective androgen receptor modulator andarine (S‐4) for routine doping control purposes

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.

Annual banned‐substance review: analytical approaches in human sports drug testing

The annual update of the list of prohibited substances and doping methods as issued by the World Anti-Doping Agency (WADA) allows the implementation of most recent considerations of performance manipulation and emerging therapeutics into human sports doping control programmes. The annual banned-substance review for human doping controls critically summarizes recent innovations in analytical approaches that support the efforts of convicting cheating athletes by improved or newly established methods that focus on known as well as newly outlawed substances and doping methods. In the current review, literature published between October 2008 and September 2009 reporting on new and/or enhanced procedures and techniques for doping analysis, as well as aspects relevant to the doping control arena, was considered to complement the 2009 annual banned-substance review.

Mass Spectrometry-Based Characterization of New Drugs and Methods of Performance Manipulation in Doping Control Analysis

Efficient and comprehensive sports drug testing necessitates frequent updating and proactive, preventive anti-doping research, and the early implementation of new, emerging drugs into routine doping controls is an essential aspect. Several new drugs and drug candidates with potential for abuse, including so-called Rycals (ryanodine receptor calstabin complex stabilizers, for example, S-107), hypoxia-inducible factor (HIF) stabilizers, and peroxisome-proliferator-activated receptor (PPAR) delta agonists (for example, GW1516), were studied using different mass spectrometry- and ion mobility-based approaches, and their gas phase dissociation behaviors were elucidated. The detailed knowledge of fragmentation routes allows a more rapid identification of metabolites and structurally related, presumably "tailor-made", analogs potentially designed for doping purposes. The utility of product ion characterization is demonstrated in particular with GW1516, for which oxidation products were readily identified in urine samples by means of diagnostic fragment ions as measured using high resolution/high accuracy mass spectrometry and higher energy collision-induced dissociation (HCD).

Characterization of two major urinary metabolites of the PPARδ-agonist GW1516 and implementation of the drug in routine doping controls

Since January 2009, the list of prohibited substances and methods of doping as established by the World Anti-Doping Agency includes new therapeutics such as the peroxisome-proliferator-activated receptor (PPAR)-delta agonist GW1516, which is categorized as a gene doping substance. GW1516 has completed phase II and IV clinical trials regarding dyslipidemia and the regulation of the lipoprotein transport in metabolic syndrome conditions; however, its potential to also improve athletic performance due to the upregulation of genes associated with oxidative metabolism and a modified substrate preference that shifted from carbohydrate to lipid consumption has led to a ban of this compound in elite sport. In a recent report, two presumably mono-oxygenated and bisoxygenated urinary metabolites of GW1516 were presented, which could serve as target analytes for doping control purposes after full characterization. Hence, in the present study, phase I metabolism was simulated by in vitro assays employing human liver microsomal fractions yielding the same oxygenation products, followed by chemical synthesis of the assumed structures of the two abundant metabolic reaction products. These allowed the identification and characterization of mono-oxygenated and bisoxygenated metabolites (sulfoxide and sulfone, respectively) as supported by high-resolution/high-accuracy mass spectrometry with higher-energy collision-induced dissociation, tandem mass spectrometry, and nuclear magnetic resonance spectroscopy. Since urine samples have been the preferred matrix for doping control purposes, a method to detect the new target GW1516 in sports drug testing samples was developed in accordance to conventional screening procedures based on enzymatic hydrolysis and liquid-liquid extraction followed by liquid chromatography, electrospray ionization, and tandem mass spectrometry. Validation was performed for specificity, limit of detection (0.1 ng/ml), recovery (72%), intraday and interday precisions (7.7-15.1%), and ion suppression/enhancement effects (<10%).

Application of FAIMS to anabolic androgenic steroids in sport drug testing

Mass spectrometric identification of anabolic androgenic steroids challenges standard doping-control methods. To reveal a doping offence the presence of prohibited anabolic androgenic steroids at trace levels in the picogram-per-millilitre range must be confirmed as reliable. Human urine samples containing epitrenbolone, metandienone metabolite (17beta -hydroxymethyl-17alpha-methyl-18-norandrost-1,4,13-trien-3-one), stanozolol, 16beta-hydroxystanozolol and 4beta-hydroxystanozolol were analysed using LC-FAIMS-MS/MS. These substances are prohibited in sport according to World Anti-Doping Agency (WADA) regulations. Glucuronides were hydrolysed and prepared by liquid-liquid extraction. Excellent recovery and precision were obtained for all compounds. Linear calibration results for epitrenbolone and metandienone metabolite were obtained and concentration information could be determined in the ranges of reliable response between 750-1200 and 100-600 pg/mL, respectively. Limits of detection were estimated at 25 pg/mL (stanozolol), 50 pg/mL (metandienone metabolite, 16beta-hydroxystanozolol), 100 pg/mL (4beta-hydroxystanozolol) and 500 pg/mL (epitrenbolone). The assay was applied to doping-control samples. For all analytes, LC-FAIMS-MS/MS resulted in excellent interference removal, which effectively extends the post-dose detection time.