New analytical methodologies for doping control – detection of anabolic androgenic steroids in human urine

Dissertation submitted to Faculdade de Ciências e Tecnologia - Universidade Nova de Lisboa in fulfilment of the requirements for the degree of Doctor of Philosophy (Biochemistry - Biotechnology)

Fast screening and confirmation of doping agents by UHPLC-QTOF-MS/MS

Introduction: The general strategy to perform anti-doping analysis starts with a screening followed by a confirmatory step when a sample is suspected to be positive. The screening step should be fast, generic and able to highlight any sample that may contain a prohibited substance by avoiding false negative and reducing false positive results. The confirmatory step is a dedicated procedure comprising a selective sample preparation and detection mode. Aim: The purpose of the study is to develop rapid screening and selective confirmatory strategies to detect and identify 103 doping agents in urine. Methods: For the screening, urine samples were simply diluted by a factor 2 with ultra-pure water and directly injected ("dilute and shoot") in the ultrahigh- pressure liquid chromatography (UHPLC). The UHPLC separation was performed in two gradients (ESI positive and negative) from 5/95 to 95/5% of MeCN/Water containing 0.1% formic acid. The gradient analysis time is 9 min including 3 min reequilibration. Analytes detection was performed in full scan mode on a quadrupole time-of-flight (QTOF) mass spectrometer by acquiring the exact mass of the protonated (ESI positive) or deprotonated (ESI negative) molecular ion. For the confirmatory analysis, urine samples were extracted on SPE 96-well plate with mixed-mode cation (MCX) for basic and neutral compounds or anion exchange (MAX) sorbents for acidic molecules. The analytes were eluted in 3 min (including 1.5 min reequilibration) with a S1-25 Ann Toxicol Anal. 2009; 21(S1) Abstracts gradient from 5/95 to 95/5% of MeCN/Water containing 0.1% formic acid. Analytes confirmation was performed in MS and MS/MS mode on a QTOF mass spectrometer. Results: In the screening and confirmatory analysis, basic and neutral analytes were analysed in the positive ESI mode, whereas acidic compounds were analysed in the negative mode. The analyte identification was based on retention time (tR) and exact mass measurement. "Dilute and shoot" was used as a generic sample treatment in the screening procedure, but matrix effect (e.g., ion suppression) cannot be avoided. However, the sensitivity was sufficient for all analytes to reach the minimal required performance limit (MRPL) required by the World Anti Doping Agency (WADA). To avoid time-consuming confirmatory analysis of false positive samples, a pre-confirmatory step was added. It consists of the sample re-injection, the acquisition of MS/MS spectra and the comparison to reference material. For the confirmatory analysis, urine samples were extracted by SPE allowing a pre-concentration of the analyte. A fast chromatographic separation was developed as a single analyte has to be confirmed. A dedicated QTOF-MS and MS/MS acquisition was performed to acquire within the same run a parallel scanning of two functions. Low collision energy was applied in the first channel to obtain the protonated molecular ion (QTOF-MS), while dedicated collision energy was set in the second channel to obtain fragmented ions (QTOF-MS/MS). Enough identification points were obtained to compare the spectra with reference material and negative urine sample. Finally, the entire process was validated and matrix effects quantified. Conclusion: Thanks to the coupling of UHPLC with the QTOF mass spectrometer, high tR repeatability, sensitivity, mass accuracy and mass resolution over a broad mass range were obtained. The method was sensitive, robust and reliable enough to detect and identify doping agents in urine. Keywords: screening, confirmatory analysis, UHPLC, QTOF, doping agents

A forensic perspective of the AFL investigation into peptides: an antidoping investigation case study.

BACKGROUND: The World Anti-Doping Agency (WADA) is introducing enhancements to doping investigations in its 2015 Code, which include improved sharing of information between antidoping organisations (including sporting bodies) and enhanced accountability of athlete support staff. These additions will improve the control of links between sports doping and organised crime. In February 2013 the Australian Crime Commission released a report that linked several professional sporting codes, professional athletes with links to organised crime, performance enhancing drugs and illicit substances. Following this report the Australian Football League (AFL) partnered the Australian national antidoping organisation to investigate peptide use in Australian football. METHODS: This review compared the model proposed by Marclay, a hypothetical model for anti-doping investigations that proposed a forensic intelligence and analysis approach, to use the forensic capabilities of the AFL investigation to test the model's relevance to an actual case. RESULTS: The investigation uncovered the use of peptides used to enhance athlete performance. The AFL investigation found a high risk of doping where athlete support staff existed in teams with weak corporate governance controls. A further finding included the need for the investigation to provide a timely response in professional team sports that were sensitive to the competition timing. In the case of the AFL the team was sanctioned prior to the finals as an interim outcome for allowing the risk of use of performance-enhancing substances. Doping violation charges are still being considered. DISCUSSION: Antidoping strategies should include the investigation of corporate officers in team doping circumstances, the mandatory recording of all athlete substance use during competition and training phases, the wider sharing of forensic intelligence with non-sporting bodies particularly law enforcement and collaboration between antidoping and sporting organisations in doping investigations. CONCLUSIONS: The AFL investigation illustrated the importance of the 2015 WADA Code changes and highlighted the need for a systematic use of broad forensic intelligence activities in the investigation of doping violations.

Evaluation of two immunoassays for the measurement of human chorionic gonadotropin in urine for anti-doping purposes.

Background: Urinary human chorionic gonadotropin (hCG) concentration is routinely measured in all anti-doping laboratories to exclude the misuse of recombinant or urinary hCG preparations. In this study, extended validation of two commercial immunoassays for hCG measurements in urine was performed. Both tests were initially designed for hCG determination in human serum/plasma. Methods: Access (R) and Elecsys (R) 1010 are two automated immunoanalysers for central laboratories. The limits of detection and quantification, as well as intra-laboratory and inter-technique correlation, precision, and accuracy, were determined. Stability studies of hCG in urine following freezing and thawing cycles (n = 3) as well as storage conditions at room temperature, 4 degrees C and 20 degrees C, were performed. Results: Statistical evaluation of hCG concentrations in male urine samples (n = 2429) measured with the Elecsys (R) 1010 system enabled us to draw a skewed frequency histogram and establish a far outside value equal to 2.3 IU/L. This decision limit corresponds to the concentration at which a sportsman will be considered positive for hCG. Intra-assay precision for the Access (R) analyser was less than 4.0 A, whereas the inter-assay precision was closer to 4.5 % (concentrations of the official external controls contained between 5.5 and 195.0 IU/L). Intra and inter-assay precision for the Elecsys (R) 1010 analyser was slightly better. A good inter-technique correlation was obtained when measuring various urine samples (male and female). No urinary hCG loss was observed after two freeze/thaw cycles. On the other hand, time and inappropriate storage conditions, such as temperatures above 10 degrees C for more than 5 days, can deteriorate urinary hCG. Conclusions: Both analysers showed acceptable performances and are suitable for screening urine for anti-doping analyses. Each laboratory should validate and establish its own reference values because hCG concentrations measured in urine can be different from one immunoassay to another. The time delay between urine collection and analysis should be reduced as much as possible, and urine samples should be transported in optimal conditions to avoid a loss of hCG immunoreactivity.

Fast analysis of doping agents in urine by ultra-high-pressure liquid chromatography-quadrupole time-of-flight mass spectrometry. II: Confirmatory analysis.

For doping control, analyses of samples are generally achieved in two steps: a rapid screening and, in the case of a positive result, a confirmatory analysis. A two-step methodology based on ultra-high-pressure liquid chromatography coupled to a quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) was developed to screen and confirm 103 doping agents from various classes (e.g., beta-blockers, stimulants, diuretics, and narcotics). The screening method was presented in a previous article as part I (i.e., Fast analysis of doping agents in urine by ultra-high-pressure liquid chromatography-quadrupole time-of-flight mass spectrometry. Part I: screening analysis). For the confirmatory method, basic, neutral and acidic compounds were extracted by a dedicated solid-phase extraction (SPE) in a 96-well plate format and detected by MS in the tandem mode to obtain precursor and characteristic product ions. The mass accuracy and the elemental composition of precursor and product ions were used for compound identification. After validation including matrix effect determination, the method was considered reliable to confirm suspect results without ambiguity according to the positivity criteria established by the World Anti-Doping Agency (WADA). Moreover, an isocratic method was developed to separate ephedrine from its isomer pseudoephedrine and cathine from phenylpropanolamine in a single run, what allowed their direct quantification in urine.

Methods for detection and confirmation of Hematide?/peginesatide in anti-doping samples.

Since the 1990's, cheating athletes have abused substances to increase their oxygen transport capabilities; among these substances, recombinant EPO is the most well known. Currently, other investigational pharmaceutical products are able to produce an effect similar to EPO but without having chemical structures related to EPO; these are the synthetic erythropoiesis stimulating agents (ESAs). Peginesatide (also known as Hematide?) is being developed by Affymax and Takeda and, if approved by regulatory authorities, could soon be released on the international market. To detect potential athletic abuse of this product and deter athletes who consider cheating, we initiated a collaboration to implement a detection test for anti-doping purposes. Peginesatide is a synthetic, PEGylated, investigational, peptide-based erythropoiesis-stimulating agent that is designed and engineered to stimulate specifically the erythropoietin receptor dimer that governs erythropoiesis. It is undetectable using current anti-doping tests due to its lack of sequence homology to EPO. To detect and deter potential abuse of peginesatide, we initiated an industry/antidoping laboratory collaboration to develop and validate screening and confirmation assays so that they would be available before peginesatide reaches the market. We describe a screening ELISA and a confirmation assay consisting of immune-purification followed by separation with SDS-PAGE and revelation with Western double blotting. Both assays can detect 0.5 ng/mL concentrations of peginesatide in blood samples, enabling detection for several days after administration of a physiologically relevant dose. This initial report describes experimental characterization of these assays, including testing with a blinded set of samples from a clinical study conducted in healthy volunteers.

Perspectives for Forensic Intelligence in anti-doping: thinking outside the box

Today's approach to anti-doping is mostly centered on the judicial process, despite pursuing a further goal in the detection, reduction, solving and/or prevention of doping. Similarly to decision-making in the area of law enforcement feeding on Forensic Intelligence, anti-doping might significantly benefit from a more extensive gathering of knowledge. Forensic Intelligence might bring a broader logical dimension to the interpretation of data on doping activities for a more future-oriented and comprehensive approach instead of the traditional case-based and reactive process. Information coming from a variety of sources related to doping, whether directly or potentially, would feed an organized memory to provide real time intelligence on the size, seriousness and evolution of the phenomenon. Due to the complexity of doping, integrating analytical chemical results and longitudinal monitoring of biomarkers with physiological, epidemiological, sociological or circumstantial information might provide a logical framework enabling fit for purpose decision-making. Therefore, Anti-Doping Intelligence might prove efficient at providing a more proactive response to any potential or emerging doping phenomenon or to address existing problems with innovative actions or/and policies. This approach might prove useful to detect, neutralize, disrupt and/or prevent organized doping or the trafficking of doping agents, as well as helping to refine the targeting of athletes or teams. In addition, such an intelligence-led methodology would serve to address doping offenses in the absence of adverse analytical chemical evidence.

Urinary analysis of 16(5alpha)-androsten-3alpha-ol by gas chromatography/combustion/isotope ratio mass spectrometry: implications in anti-doping analysis.

We present a method for the analysis of urinary 16(5alpha)-androsten-3alpha-ol together with 5beta-pregnane-3alpha,20alpha-diol and four testosterone metabolites: androsterone (Andro), etiocholanolone (Etio), 5alpha-androstane-3alpha,17beta-diol (5alphaA), 5beta-androstane-3alpha,17beta-diol (5betaA) by means of gas chromatography/combustion/isotopic ratio mass spectrometry (GC/C/IRMS). The within-assay and between-assay precision S.D.s of the investigated steroids were lower than 0.3 and 0.6 per thousand, respectively. A comparative study on a population composed of 20 subjects has shown that the differences of the intra-individual delta(13)C-values for 16(5alpha)-androsten-3alpha-ol and 5beta-pregnane-3alpha,20alpha-diol are less than 0.9 per thousand. Thereafter, the method has been applied in the frame of an excretion study following oral ingestion of 50 mg DHEA initially and oral ingestion of 50mg pregnenolone 48 h later. Our findings show that administration of DHEA does not affect the isotopic ratio values of 16(5alpha)-androsten-3alpha-ol and 5beta-pregnane-3alpha,20alpha-diol, whereas the isotopic ratio values of 5beta-pregnane-3alpha,20alpha-diol vary by more 5 per thousand upon ingestion of pregnenolone. We have observed delta(13)C-value changes lower than 1 per thousand for 16(5alpha)-androsten-3alpha-ol, though pregnenolone is a precursor of the 16-ene steroids. In contrast to 5beta-pregnane-3alpha,20alpha-diol, the 16-ene steroid may be used as an endogenous reference compound when pregnenolone is administered.

Detection of erythropoiesis-stimulating agents in human anti-doping control: past, present and future.

Stimulation of erythropoiesis is one of the most efficient ways of doping. This type of doping is advantageous for aerobic physical exercise and of particular interest to endurance athletes. Erythropoiesis, which takes place in bone marrow, is under the control of EPO, a hormone secreted primarily by the kidneys when the arterial oxygen tension decreases. In certain pathological disorders, such as chronic renal failure, the production of EPO is insufficient and results in anemia. The pharmaceutical industry has, thus, been very interested in developing drugs that stimulate erythropoiesis. With this aim, various strategies have been, and continue to be, envisaged, giving rise to an expanding range of drugs that are good candidates for doping. Anti-doping control has had to deal with this situation by developing appropriate methods for their detection. This article presents an overview of both the drugs and the corresponding methods of detection, and thus follows a roughly chronological order.

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. To screen for misuse with these compounds, drug testing laboratories monitor the urinary concentrations of endogenous steroid metabolites and their ratios, which constitute the steroid profile and compare them with reference ranges to detect unnaturally high values. However, the interpretation of the steroid profile is difficult due to large inter-individual variances, various confounding factors and different endogenous steroids marketed that influence the steroid profile in various ways. A support vector machine (SVM) algorithm was developed to statistically evaluate urinary steroid profiles composed of an extended range of steroid profile metabolites. This model makes the interpretation of the analytical data in the quest for deviating steroid profiles feasible and shows its versatility towards different kinds of misused endogenous steroids. The SVM model outperforms the current biomarkers with respect to detection sensitivity and accuracy, particularly when it is coupled to individual data as stored in the Athlete Biological Passport.

Short term impact of Tribulus terrestris intake on doping control analysis of endogenous steroids.

Tribulus terrestris is a nutritional supplement highly debated regarding its physiological and actual effects on the organism. The main claimed effect is an increase of testosterone anabolic and androgenic action through the activation of endogenous testosterone production. Even if this biological pathway is not entirely proven, T. terrestris is regularly used by athletes. Recently, the analysis of two female urine samples by GC/C/IRMS (gas chromatography/combustion/isotope-ratio-mass-spectrometry) conclusively revealed the administration of exogenous testosterone or its precursors, even if the testosterone glucuronide/epitestosterone glucuronide (T/E) ratio and steroid marker concentrations were below the cut-off values defined by World Anti-Doping Agency (WADA). To argue against this adverse analytical finding, the athletes recognized having used T. terrestris in their diet. In order to test this hypothesis, two female volunteers ingested 500 mg of T. terrestris, three times a day and for two consecutive days. All spot urines were collected during 48 h after the first intake. The (13)C/(12)C ratio of ketosteroids was determined by GC/C/IRMS, the T/E ratio and DHEA concentrations were measured by GC/MS and LH concentrations by radioimmunoassay. None of these parameters revealed a significant variation or increased above the WADA cut-off limits. Hence, the short-term treatment with T. terrestris showed no impact on the endogenous testosterone metabolism of the two subjects.

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.

Smoked cannabis and doping control: looking for the wrong target analyte?

Introduction: Since 2004, cannabis is prohibited by the World Anti-Doping Agency (WADA) for all sports in competition. In the years since then, about half of all positive doping cases in Switzerland have been related to cannabis consumption. In most cases, the athletes plausibly claim to have consumed cannabis several days or even weeks before competition and only for recreational purposes not related to competition. In doping analysis, the target analyte in urine samples is 11-nor-delta-9-tetrahydrocannabinol- 9-carboxylic acid (THC-COOH), the reporting threshold for laboratories is 15 ng/mL. However, the wide detection window of this long-term THC metabolite in urine does not allow a conclusion concerning the time of consumption or the impact on the physical performance. Aim: The purpose of the present pharmacokinetic study on volunteers was to evaluate target analytes with shorter urinary excretion time. Subsequently, urines from athletes tested positive for cannabis should be reanalyzed including these analytes. Methods: In an one-session clinical trial (approved by IRB, Swissmedic, and Federal Office of Public Health), 12 healthy, male volunteers (age 26 ± 3 yrs, BMI 24 ± 2 kg/m2) with cannabis experience (> once/month) smoked a Cannabis cigarette standardized to 70 mg THC/cigarette (Bedrobinol® 7%, Dutch Office for Medicinal Cannabis) following a paced-puffing procedure. Plasma and urine was collected up to 8 h and 11 days, respectively. Total THC, 11-hydroxy-THC (THC-OH), and THC-COOH were determined after enzymatic hydrolyzation followed by SPE and GC/MS-SIM. The limit of quantitation (LOQ) for all analytes was 0.1 ng/mL. Visual analog scales (VAS) and vital functions were used for monitoring psychological and somatic side-effects at every timepoint of specimen collection (up to 480 min). Results: Eight puffs delivered a mean THC dose of 45 mg. Mean plasma levels of total THC, THC-OH and THC-COOH were measured in the range of 0.1-20.9, 0.1-1.8, and 1.8-7.5 ng/mL, respectively. Peak concentrations were observed at 5, 10, and 90 min. Mean urine levels were measured in the range of 0.1-0.7, 0.10-6.2, and 0.1-13.4 ng/mL, respectively. The detection windows were 2-8, 2-96, and 2-120 h. No or only mild effects were observed, such as dry mouth, sedation, and tachycardia. Besides high to very high THC-COOH levels (0-978 ng/mL), THC (0.1-24 ng/mL) and THC-OH (1-234 ng/mL) were found in 90 and 96% of the cannabis-positive urines from athletes. Conclusion: Instead of or in addition to THC-COOH, the pharmacologically active THC and THC-OH should be the target analytes for doping urine analysis. This would allow the estimation of more recent Cannabis consumption, probably influencing performance during competition. Keywords: cannabis, doping, clinical trial, plasma and urine levels, athlete's samples