New and old challenges of sports drug testing

This brief note gives a general overview on the activity of the antidoping laboratories accredited by the World Anti-Doping Agency (WADA), outlining the evolution, over the last four decades, of the analytical methods and techniques in the detection of prohibited substances and methods. Special emphasis is given to the future trends of the fight against doping in sports, as seen from the perspective of a laboratory scientist, in the wider context of fair play, health protection, and perception of the activity of the antidoping laboratories by the general public.     

Statistical discrimination of steroid profiles in doping control with support vector machines

Due to their performance enhancing properties, use of anabolic steroids (e.g. testosterone, nandrolone, etc.) is banned in elite sports. Therefore, doping control laboratories accredited by the World Anti-Doping Agency (WADA) screen among others for these prohibited substances in urine. It is particularly challenging to detect misuse with naturally occurring anabolic steroids such as testosterone (T), which is a popular ergogenic agent in sports and society.     

The application of carbon isotope ratio mass spectrometry to doping control

The administration of synthetic steroid copies is one of the most important issues facing sports. Doping control laboratories accredited by the World Anti-Doping Agency (WADA) require methods of analysis that allow endogenous steroids to be distinguished from their synthetic analogs in urine. The ability to measure isotope distribution at natural abundance with high accuracy and precision has increased the application of Gas Chromatography-Combustion-Isotope Ratio Mass Spectrometry (GC-C-IRMS) to doping control in recent years. GC-C-IRMS is capable of measuring the carbon isotope ratio (delta(13)C) of urinary steroids and confirm their synthetic origin based on the abnormal (13)C content. This tutorial describes some of the complexities encountered by obtaining valid delta(13)C measurements from GC-C-IRMS and the need for careful interpretation of all relevant information concerning an individual's metabolism in order to make an informed decision with respect to a doping violation.     

Evaluation of World Anti-Doping Agency criteria for anabolic agent analysis by using comprehensive two-dimensional gas chromatography–mass spectrometry

This work presents the validation study of the comprehensive two-dimensional gas chromatography (GC×GC)–time-of-flight mass spectrometry method performance in the analysis of the key World Anti-Doping Agency (WADA) anabolic agents in doping control. The relative abundance ratio, retention time, identification and other method performance criteria have been tested in the GC×GC format to confirm that they comply with those set by WADA. Furthermore, tens of other components were identified with an average similarity of >920 (on the 0–999 scale), including 10 other endogenous sterols, and full mass spectra of 5,000+ compounds were retained. The testosterone/epitestosterone ratio was obtained from the same run. A new dimension in doping analysis has been implemented by addressing separation improvement. Instead of increasing the method sensitivity, which is accompanied by making the detector increasingly “blind” to the matrix (as represented by selected ion monitoring mode, high-resolution mass spectrometry (MS) and tandem MS), the method capabilities have been improved by adding a new “separation” dimension while retaining full mass spectral scan information. Apart from the requirement for the mass spectral domain that a minimum of three diagnostic ions with relative abundance of 5% or higher in the MS spectra, all other WADA criteria are satisfied by GC×GC operation. The minimum of three diagnostic ions arises from the need to add some degree of specificity to the acquired mass spectrometry data; however, under the proposed full MS scan method, the high MS similarity to the reference compounds offers more than the required three diagnostic ions for an unambiguous identification. This should be viewed as an extension of the present criteria to a full-scan MS method.     

Validation of a GC/MS method for the detection of two quinolinone-derived selective androgen receptor modulators in doping control analysis

Selective androgen receptor modulators (SARMs) represent an emerging class of drugs likely to be abused in sport. For clinical applications, these substances provide a promising alternative to testosterone-replacement therapies and their advantages include oral bioavailability, androgen receptor specificity, tissue selectivity, and the absence of steroid-related side effects. Although not yet commercially available, since January 2008 SARMs have been included on the prohibited list issued yearly by the World Anti-Doping Agency (WADA), so control laboratories need to update their procedures to detect either the parent drugs or their metabolites. Within this context, two quinolinone SARM models were synthesized and automatically characterized to update the existing routine screening procedures. The conditions for the new target analytes are compatible with the existing laboratory protocols used for both in-competition and out-of-competition controls and can be included in them. Validation parameters according to ISO 17025 and WADA guidelines were successfully determined. For analytical determinations, spiked urine samples were hydrolyzed and extracted at pH 9.6 with 10 mL of tert-butyl methyl ether. Then, the analytes were subsequently converted into trimethylsilyl derivatives and detected by gas chromatography–mass spectrometry. The absence of interferents, together with excellent repeatability of both retention times and the relative abundances of diagnostic ions, allowed proper identification of all SARM analytes. The analytes’ quantification was linear up to 500 ng/mL and precision criteria were satisfied (coefficient of variation less than 25% at 10 ng/mL). The limits of detection were 1 ng/mL for both SARMs, whereas recovery values were between 95.5 and 99.3%. The validated method can be efficiently used for urine screening of the 2-quinolinone-derived SARMs tested.     

Chemoinformatics-based classification of prohibited substances employed for doping in sport

Representative molecules from 10 classes of prohibited substances were taken from the World Anti-Doping Agency (WADA) list, augmented by molecules from corresponding activity classes found in the MDDR database. Together with some explicitly allowed compounds, these formed a set of 5245 molecules. Five types of fingerprints were calculated for these substances. The random forest classification method was used to predict membership of each prohibited class on the basis of each type of fingerprint, using 5-fold cross-validation. We also used a k-nearest neighbors (kNN) approach, which worked well for the smallest values of k. The most successful classifiers are based on Unity 2D fingerprints and give very similar Matthews correlation coefficients of 0.836 (kNN) and 0.829 (random forest). The kNN classifiers tend to give a higher recall of positives at the expense of lower precision. A na?ve Bayesian classifier, however, lies much further toward the extreme of high recall and low precision. Our results suggest that it will be possible to produce a reliable and quantitative assignment of membership or otherwise of each class of prohibited substances. This should aid the fight against the use of bioactive novel compounds as doping agents, while also protecting athletes against unjust disqualification.     

An international intercomparison for 19-norandrosterone in human urine: the Comité Consultatif pour la Quantité de Matière (CCQM) Pilot Study CCQM-P68

Under the auspices of the Comité Consultatif pour la Quantité de Matière (CCQM) a laboratory intercomparison, CCQM-P68, was co-ordinated by the National Measurement Institute of Australia (NMIA) in 2005 involving the analysis of the steroid 19-norandrosterone (19-NA) in human urine. 19-NA is the major metabolite of nandrolone, a commonly abused anabolic agent. It is routinely tested by many sports drug testing laboratories and several national metrology institutes have developed reference methods to provide reference values for the certification of reference materials. The material distributed for the intercomparison was a freeze-dried human urine candidate reference material containing 19-NA at a level close to the allowed World Anti-Doping Agency (WADA) cut-off for this anabolic agent, i.e. 2 ng/ml. Four national/designated metrology institutes participated in the intercomparison and their results for this complex analysis were in excellent agreement, with a relative standard deviation (RSD) of only 1.7%. The expanded uncertainties at the 95% level of confidence for the reported results ranged from 3.7 to 7.0% at the 2.15 ng/g level.     

Quantification and stability of salbutamol in human urine

A sensitive method for the quantification of free salbutamol in human urine is described. Sample clean up is performed using SPE on a mixed phase extraction column. Derivatisation is performed with N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) and the extract is analysed by GC-MS. The method was found to be suitable for use in the doping field, where a cut-off limit of 1 microg salbutamol/mL urine is set by the International Olympic Committee (IOC) and approved by the World Anti-Doping Agency (WADA). Above that value a doping violation occurs. In addition, the stability of salbutamol in human urine has been evaluated.     

Simultaneous analysis of fourteen tertiary amine stimulants in human urine for doping control purposes by liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry

A method for the simultaneous screening and confirmation of the presence of fourteen tertiary amine stimulants in human urine by gas chromatography-mass spectrometry (GC-MS) in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed and validated. Solid phase extraction (SPE) and liquid-liquid extraction (LLE) approaches were utilized for the pre-treatment of the urine samples. The study indicated that the capillary temperature played a significant role in the signal abundances of the protonated molecules of cropropamide and crotethamide under positive ion electrospray ionization (ESI) conditions. In addition, comparison studies of two different pre-treatment approaches as well as the two ionization modes were conducted. The LODs of the developed method for all the analytes were lower than the minimum required performance limit (MRPL) as set forth in the World Anti-Doping Agency (WADA) technical document for laboratories. The human urine sample obtained after oral administration of prolintane·HCl was successfully analyzed by the developed method, which demonstrated the applicability and reliability of the method for routine doping control analysis.     

Synthesis and identification of hydroxylated metabolites of the anti-estrogenic agent cyclofenil

The detection of metabolites of the anti-estrogenic substance cyclofenil, listed on the World Anti-Doping Agency (WADA) Prohibited List since 2004 is described. Target substances are hydroxylated metabolites, bearing an aliphatic hydroxyl group either in the 2-, 3- or 4-position of the aliphatic ring, in addition to the phenolic functions on the aromatic rings. Structural identification used NMR as well as high-resolution mass spectrometry after nano-electrospray ionisation (ESI). Unambiguous detection of all three synthesised cyclofenil metabolites M1-M3 was done using gas chromatography for separation and electron ionisation mass spectrometry for detection of the per-silylated compounds in comparison with a reference urine deriving from an excretion study within the WADA 2007 Educational Programme.     

Use of isotope ratio mass spectrometry to differentiate between endogenous steroids and synthetic homologues in cattle: A review

Although substantial technical advances have been achieved during the past decades to extend and facilitate the analysis of growth promoters in cattle, the detection of abuse of synthetic analogs of naturally occurring hormones has remained a challenging issue. When it became clear that the exogenous origin of steroid hormones could be traced based on the ¹³C/¹²C isotope ratio of the substances, GC/C/IRMS has been successfully implemented to this aim since the end of the past century. However, due to the costly character of the instrumental setup, the susceptibility of the equipment to errors and the complex and time consuming sample preparation, this method is up until now only applied by a limited number of laboratories. In this review, the general principles as well as the practical application of GC/C/IRMS to differentiate between endogenous steroids and exogenously synthesized homologous compounds in cattle will be discussed in detail, and will be placed next to other existing and to be developed methods based on isotope ratio mass spectrometry. Finally, the link will be made with the field of sports doping, where GC/C/IRMS has been established within the World Anti-Doping Agency (WADA) approved methods as the official technique to differentiate between exogenous and endogenous steroids over the past few years.     

Application of multivariate statistics to the Steroidal Module of the Athlete Biological Passport: A proof of concept study

The Technical Document TD2014EAAS was drafted by the World Anti-Doping Agency (WADA) in order to fight the spread of endogenous anabolic androgenic steroids (EAAS) misuse in several sport disciplines. In particular, adoption of the so-called Athlete Biological Passport (ABP) – Steroidal Module allowed control laboratories to identify anomalous EAAS concentrations within the athletes' physiological urinary steroidal profile. Gas chromatography (GC) combined with mass spectrometry (MS), indicated by WADA as an appropriate technique to detect urinary EAAS, was utilized in the present study to develop and fully-validate an analytical method for the determination of all EAAS markers specified in TD2014EAAS, plus two further markers hypothetically useful to reveal microbial degradation of the sample. In particular, testosterone, epitestosterone, androsterone, etiocholanolone, 5α-androstane-3α,17β-diol, 5β-androstane-3α,17β-diol, dehydroepiandrosterone, 5α-dihydrotestosterone, were included in the analytical method. Afterwards, the multi-parametric feature of ABP profile was exploited to develop a robust approach for the detection of EAAS misuse, based on multivariate statistical analysis. In particular, Principal Component Analysis (PCA) was combined with Hotelling T² tests to explore the EAAS data obtained from 60 sequential urine samples collected from six volunteers, in comparison with a reference population of single urine samples collected from 96 volunteers. The new approach proved capable of identifying anomalous results, including (i) the recognition of samples extraneous to each of the individual urine series and (ii) the discrimination of the urine samples collected from individuals to whom “endogenous” steroids had been administrated with respect to the rest of the samples population. The proof-of-concept results presented in this study will need further extension and validation on a population of sport professionals.     

Introduction of solid-phase microextraction as a high-throughput sample preparation tool in laboratory analysis of prohibited substances

A fully automated, high-throughput method based on thin-film solid-phase microextraction (SPME) and liquid chromatography-mass spectrometry was developed for simultaneous quantitative analysis of 110 doping compounds, selected from ten classes and varying in physical and chemical properties. Among four tested extraction phases, C18 blades were chosen, as they provided optimum recoveries and the lowest carryover effect. The SPME method was optimized in terms of extraction pH, ionic strength of the sample, washing solution, extraction and desorption times for analysis of urine samples. Chromatographic separation was obtained in reversed-phase model; for detection, two mass spectrometers were used: triple quadrupole and full scan orbitrap. These combinations allowed for selective analysis of targeted compounds, as well as a retrospective study for known and unknown compounds. The developed method was validated according to the Food and Drug Administration (FDA) criteria, taking into account Minimum Required Performance Level (MRPL) values required by the World Anti-Doping Agency (WADA). In addition to analysis of free substances, it was also shown that the proposed method is able to extract the glucuronated forms of the compounds. The developed assay offers fast and reliable analysis of various prohibited substances, an attractive alternative to the standard methods that are currently used in anti-doping laboratories.     

Multiplexed immunoassay to detect anabolic androgenic steroids in human serum

A multianalyte enzyme-linked immunosorbent assay (ELISA) has been developed for the simultaneous detection of anabolic androgenic steroids (AAS) in human serum. The multiplexed method was developed according to a planar strategy in which the analytes are identified by their location in the microtiter plate. In the immunochemical procedure established here, human serum samples are mixed with a cocktail of antibodies and added to the distinct sections of a microplate biofunctionalized with different haptenized biomolecules. The cocktail of antibodies consists of a mixture of polyclonal antibodies raised against stanozolol (ST), boldenone (B), and tetrahydrogestrinone (THG). The whole immunochemical analytical procedure takes around 2 h including sample preparation, and many samples can be processed simultaneously to screen for the presence of the three AAS in a single run. Using this ELISA, ST, B, and THG can be detected and quantified individually. When used as a screening method, due to the cross-reactivity profiles of the immunoreagents used, the presence of up to 11 AAS can be detected simultaneously. The detectabilities achieved by this method in human serum are below the MRPLs (minimum required performance limits) proposed by WADA (World Anti-Doping Agency) and reference laboratories of the European Community.     

Stabilization of human urine doping control samples: a current opinion

Transportation of doping control urine samples from the collection sites to the World Anti-doping Agency (WADA) Accredited Laboratories is conducted under ambient temperatures. When sample delivery is not immediate, microbial contamination of urine, especially in summer, is a common phenomenon that may affect sample integrity and may result in misinterpretation of analytical data. Furthermore, the possibility of intentional contamination of sports samples during collection with proteolytic enzymes, masking the abuse of prohibited proteins such as erythropoietin (EPO) and peptide hormones, is a practice that has already been reported. Consequently, stabilization of urine samples with a suitable method in a way that protects samples’ integrity is important. Currently, no stabilization method is applied in the sample collection equipment system in order to prevent degradation of urine compounds. The present work is an overview of a study, funded by WADA, on degradation and stabilization aspects of sports urine samples against the above threats of degradation. Extensive method development resulted in the creation of a mixture of chemical agents for the stabilization of urine. Evaluation of results demonstrated that the stabilization mixture could stabilize endogenous steroids, recombinant EPO, and human chorionic gonadotropin in almost the entire range of the experimental conditions tested.     

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.     

Mass spectrometry of selective androgen receptor modulators

Nonsteroidal selective androgen receptor modulators (SARMs) are an emerging class of drugs for treatment of various diseases including osteoporosis and muscle wasting as well as the correction of age-related functional decline such as muscle strength and power. Several SARMs, which have advanced to preclinical and clinical trials, are composed of diverse chemical structures including arylpropionamide-, bicyclic hydantoin-, quinoline-, and tetrahydroquinoline-derived nuclei. Since January 2008, SARMs have been categorized as anabolic agents and prohibited by the World Anti-Doping Agency (WADA). Suitable detection methods for these low-molecular weight drugs were based on mass spectrometric approaches, which necessitated the elucidation of dissociation pathways in order to characterize and identify the target analytes in doping control samples as well as potential metabolic products and synthetic analogs. Fragmentation patterns of representatives of each category of SARMs after electrospray ionization (ESI) and collision-induced dissociation (CID) as well as electron ionization (EI) are summarized. The complexity and structural heterogeneity of these drugs is a daunting challenge for detection methods.     

Chromatographic–mass spectrometric analysis of peptidic analytes (2–10 kDa) in doping control urine samples

Peptides with a molecular mass between 2 and 10 kDa that are prohibited in elite sports usually require dedicated sample preparation and mass spectrometric detection that commonly cannot be combined with other (lower molecular mass) substances. In most instances, the physicochemical differences are too significant to allow for a generic analytical procedure. A simplification of established and comparably complex analytical approaches is therefore desirable and has been accomplished in the context of this study. With urine samples representing still the most frequently collected doping control specimens, efficient extraction of peptidic analytes from this matrix was a major goal of this method, as demonstrated for the included compounds such as insulins (human, lispro, aspart, glulisine, tresiba, glargine metabolite, bovine insulin, porcine insulin), growth hormone-releasing hormones (sermorelin, CJC-1295, tesamorelin) incl. their respective metabolites, insulin-like-growth factors (long-R₃-IGF-I, R₃-IGF-I, des_1–3-IGF-I), synacthen, gonadorelin and mechano growth factors (human MGF, MGF-Goldspink). Sample preparation and detection are controlled by five internal standards, covering all five included peptide drug categories. Nearly all requirements of the recent technical documents from the World Anti-Doping Agency (WADA) considering their minimum required performance levels (MRPL) are fulfilled, and the method was validated for its utilisation as initial testing procedure in doping controls. Finally, the approach was applied to authentic post-administration study urine samples (for insulins and gonadorelin) in order to provide proof of principle.     

Stabilization of human urine doping control samples: IV. Human chorionic gonadotropin

The presence of proteolytic enzymes in urine samples, coming from exogenous or endogenous sources, enhances the cleavage of human chorionic gonadotropin (hCG). Moreover, elevated temperatures occurring occasionally during the delayed transportation of sport urine samples, favor the nicking of the hCG molecule. The aim of the current study, funded by the World Anti-Doping Agency (WADA), was the application of a stabilization mixture in athletes’ urine samples to chemically inactivate proteolytic enzymes coming from exogenous or endogenous sources so as to prevent the degradation of hCG. The stabilization mixture applied, already tested for the stabilization of endogenous steroids and recombinant erythropoietin (rEPO), was a combination of antibiotics, antimycotic substances, and protease inhibitors. Incubation experiments were conducted in the presence or absence of the stabilization mixture in urine aliquots spiked with six proteases (first series of experiments) and one microorganism associated with urinary tract infections (UTI) (second series of experiments). Intact hCG levels were evaluated by using the EIAgen Total hCG kit. In the first series of experiments, hCG levels were reduced in the untreated aliquots following incubation at 37 °C. The addition of the chemical stabilization mixture prevented degradation of hCG induced by four of the proteases applied. In the second series of experiments, no significant difference was found in urine inoculated with E. coli, between aliquots treated with chemical mixture and the untreated aliquots. The addition of the proposed chemical stabilization mixture improves the quality of athletes’ urine samples against possible deterioration due to high temperatures or attempts of proteolytic manipulation.     

Determination of Vasopressin and Desmopressin in urine by means of liquid chromatography coupled to quadrupole time-of-flight mass spectrometry for doping control purposes

The anti-diuretic neurohypophysial hormone Vasopressin (Vp) and its synthetic analogue Desmopressin (Dp, 1-desamino-vasopressin) have received considerable attention from doping control authorities due to their impact on physiological blood parameters. Accordingly, the illicit use of Desmopressin in elite sport is sanctioned by the World Anti-Doping Agency (WADA) and the drug is classified as masking agent. Vp and Dp are small (8-9 amino acids) peptides administered orally as well as intranasally. Within the present study a method to determine Dp and Vp in urinary doping control samples by means of liquid chromatography coupled to quadrupole high resolution time-of-flight mass spectrometry was developed. After addition of Lys-Vasopressin as internal standard and efficient sample clean up with a mixed mode solid phase extraction (weak cation exchange), the samples were directly injected into the LC-MS system. The method was validated considering the parameters specificity, linearity, recovery (80-100%), accuracy, robustness, limit of detection/quantification (20/50 pg mL(-1)), precision (inter/intra-day<10%), ion suppression and stability. The analysis of administration study urine samples collected after a single intranasal or oral application of Dp yielded in detection windows for the unchanged target analyte for up to 20 h at concentrations between 50 and 600 pg mL(-1). Endogenous Vp was detected in concentrations of approximately 20-200 pg mL(-1) in spontaneous urine samples obtained from healthy volunteers. The general requirements of the developed method provide the characteristics for an easy transfer to other anti-doping laboratories and support closing another potential gap for cheating athletes.