Human Doping Control Research Articles

Annual banned-substance review 16th edition-Analytical approaches in human sports drug testing 2022/2023.

In this 16th edition of the annual banned-substance review on analytical approaches in human sports drug testing, literature on recent developments in this particular section of global anti-doping efforts that was published between October 2022 and September 2023 is summarized and discussed. Most recent additions to the continuously growing portfolio of doping control analytical approaches and investigations into analytical challenges in the context of adverse analytical findings are presented, taking into account existing as well as emerging challenges in anti-doping, with specific focus on substances and methods of doping recognized in the World Anti-Doping Agency's 2023 Prohibited List. As in previous years, focus is put particularly on new or enhanced analytical options in human doping controls, appreciating the exigence and core mission of anti-doping and, equally, the conflict arising from the opposingly trending extent of the athlete's exposome and the sensitivity of instruments nowadays commonly available in anti-doping laboratories.

A multivariate data analysis approach for the investigation of in vitro derived metabolites of ACP-105 in comparison with human in vivo metabolites

Selective androgen receptor modulators (SARMs) such as ACP-105 are prohibited in sports due to their anabolic properties. ACP-105 has in previous equine studies shown to undergo extensive metabolism, which makes its metabolite profile important to investigate in humans, since the metabolism is unknown in this species. The aims of the study were to systematically optimize in vitro microsome incubations for improved metabolite yield and to utilize a multivariate data analysis (MVDA) approach to aid the metabolite discovery. Microsomes together with S9 fractions were used at optimal conditions, both with and without phase II additives. Furthermore, the relevance of the in vitro derived metabolites was evaluated as analytical targets in doping control by comparison with results from a human post-administration urine sample collected after a single dose of 100 µg ACP-105. All samples were analyzed with liquid chromatography - Orbitrap mass spectrometry.The use of the systematical optimization and MVDA greatly simplified the search and a total of 18 in vitro metabolites were tentatively identified. The yield of the two main monohydroxylated isomers increased by 24 and 10 times, respectively. In the human urine sample, a total of seven metabolites of ACP-105, formed by a combination of hydroxylations and glucuronic acid conjugations, were tentatively identified. The main metabolites were two monohydroxylated forms that are suggested as analytical targets for human doping control after hydrolysis. All the in vivo metabolites could be detected with the MVDA approach on the in vitro models, demonstrating its usefulness for prediction of the in vivo metabolite profile.

Comparison of in vitro approaches for predicting the metabolism of the selective androgen receptor modulator RAD140

The identification of metabolites allows for the expansion of possible targets for anti-doping analysis. Especially for novel substances such as selective androgen receptor modulators (SARMs), information on metabolic fate is scarce. Novel approaches such as the organ on a chip technology may provide a metabolic profile that resembles human in vivo samples more closely than approaches that rely on human liver fractions only. In this study, the SARM RAD140 was metabolized by means of subcellular human liver fractions, human liver spheroids in an organ on a chip platform, and electrochemical (EC) conversion. The resulting metabolites were analyzed with LC-HRMS/MS and compared to a human doping control urine sample that yielded an adverse analytical finding for RAD140. A total of 16 metabolites were detected in urine, while 14, 13, and 7 metabolites were detected in samples obtained from the organ on a chip experiment, the subcellular liver fraction, and EC experiments, respectively. All tested techniques resulted in the detection of RAD140 metabolites. In the organ on a chip samples, the highest number of metabolites were detected. The subcellular liver fractions and organ on a chip techniques are deemed complementary to predict metabolites of RAD140, as both techniques produce distinct metabolites that are also found in an anonymized human in vivo urine sample.Graphical abstract

Annual banned-substance review-Analytical approaches in human sports drug testing 2021/2022.

Also in 2021/2022, considerable efforts were invested into advancing human sports drug testing programs, recognizing and taking into account existing as well as emerging challenges in anti-doping, especially with regard to substances and methods of doping specified in the World Anti-Doping Agency's 2022 Prohibited List. In this edition of the annual banned-substance review, literature on recent developments published between October 2021 and September 2022 is summarized and discussed. Focus is put particularly on enhanced analytical approaches and complementary testing options in human doping controls, appreciating the exigence and mission in anti-doping and, equally, the contemporary "new normal" considering, for example, the athlete's exposome versus analytical sensitivity and applicable anti-doping regulations for result interpretation and management.

Annual banned-substance review: Analytical approaches in human sports drug testing 2020/2021.

Most core areas of anti-doping research exploit and rely on analytical chemistry, applied to studies aiming at further improving the test methods' analytical sensitivity, the assays' comprehensiveness, the interpretation of metabolic profiles and patterns, but also at facilitating the differentiation of natural/endogenous substances from structurally identical but synthetically derived compounds and comprehending the athlete's exposome. Further, a continuously growing number of advantages of complementary matrices such as dried blood spots have been identified and transferred from research to sports drug testing routine applications, with an overall gain of valuable additions to the anti-doping field. In this edition of the annual banned-substance review, literature on recent developments in anti-doping published between October 2020 and September 2021 is summarized and discussed, particularly focusing on human doping controls and potential applications of new testing strategies to substances and methods of doping specified in the World Anti-Doping Agency's 2021 Prohibited List.

A uniform sample preparation procedure for gas chromatography combustion isotope ratio mass spectrometry for all human doping control relevant anabolic steroids using online 2/3-dimensional liquid chromatography fraction collection

Androgenic anabolic steroids are the most misused substances in sports because of their performance-enhancing effects. Often synthetic analogues of endogenously present steroids are administered. To determine their endogenous or exogenous origin, Gas Chromatography Combustion Isotope Ratio Mass Spectrometry (GC-C-IRMS) is used in the field of doping control. Compounds subjected to IRMS analysis must be interference-free, with liquid chromatography fraction collection (HPLC-FC) being the crucial clean-up step. However, this clean-up is challenging, particularly for compounds present at low concentrations in samples with pronounced matrix effects. The compounds of interests for IRMS analyses in doping control are testosterone (T) and its main metabolites (androsterone, etiocholanolone, 5α-androstane-3α,17β-diol, 5β-androstane-3α,17β-diol), epitestosterone, 19-norandrosterone (19-NA), boldenone (B) and its main metabolite (BM), formestane (F) and 6αOH-androstenedione (6aOHADION). Currently, the available methods only deal with a selection of the above-mentioned compounds. Some of these compounds (e.g., 19-NA, B, BM, 6aOHADION) are present in very low concentrations, requiring an extensive and dedicated sample clean-up, and this makes it challenging to develop a universal clean-up procedure. Many of these methods require different and multiple offline HPLC-FC setups, which are labour-intensive and time-consuming. That is problematic during, e.g., large sports events, where reporting time is limited (e.g., 72 h). Therefore, in the current work, we developed a uniform online 2D/3D HPLC-FC method, capable of purifying all relevant target compounds in a single run, leading to the fastest clean-up procedure so far (i.e., 31 min for T and its main metabolites; 46 min for 19-NA, F and 6aOHADION; 48 min for B and BM).

Annual banned-substance review: Analytical approaches in human sports drug testing 2019/2020.

Analytical chemistry-based research in sports drug testing has been a dynamic endeavor for several decades, with technology-driven innovations continuously contributing to significant improvements in various regards including analytical sensitivity, comprehensiveness of target analytes, differentiation of natural/endogenous substances from structurally identical but synthetically derived compounds, assessment of alternative matrices for doping control purposes, and so forth. The resulting breadth of tools being investigated and developed by anti-doping researchers has allowed to substantially improve anti-doping programs and data interpretation in general. Additionally, these outcomes have been an extremely valuable pledge for routine doping controls during the unprecedented global health crisis that severely affected established sports drug testing strategies. In this edition of the annual banned-substance review, literature on recent developments in anti-doping published between October 2019 and September 2020 is summarized and discussed, particularly focusing on human doping controls and potential applications of new testing strategies to substances and methods of doping specified the World Anti-Doping Agency's 2020 Prohibited List.

Implementation of the HIF activator IOX-2 in routine doping controls - Pilot study data.

Early in 2020, racehorse doping cases revolved around the hypoxia-inducible factor (HIF) activator IOX-2. While the composition of IOX-2 has also been known and monitored in human doping controls for several years, the testing capability of routine sports drug testing methods was revisited for this newly surfaced doping agent. IOX-2 and the analytically well-established HIF activator roxadustat (FG-4592) share identical precursor/product ion pairs, enabling their co-detection in existing initial testing procedures in routine doping controls for the intact unconjugated analytes. In addition, hydroxylated IOX-2 and the corresponding glucuronic acid conjugates were identified as major metabolites in a microdose elimination study, contributing to enhanced initial testing and confirmation procedures.

A high throughput approach for determination of dermorphin in human urine using liquid chromatography-mass spectrometry for doping control purposes.

Dermorphin is a peptide with analgesic actions similar to morphine, but with greater effect and less potential to cause tolerance. The use of dermorphin has been documented in race horses, and its use in humans has already been reported. Considering the potential advantages from the use of dermorphin over morphine, a method to monitor it, and its main metabolite dermorphin (1-4) in humans becomes necessary for doping control. Here, we present two orthogonal methods for this purpose: a high-throughput liquid chromatography coupled to high-resolution mass spectrometry (HRMS) as an initial testing procedure and liquid chromatography-tandem mass spectrometry (MS/MS) in the selected reaction monitoring (SRM) acquisition mode for a confirmation procedure. For urine samples, pretreatment through a mixed-mode weak cation-exchange solid-phase extraction emerged as an effective approach to extract peptides from the biological sample. For the HRMS analysis, a full-MS scan acquisition mode was selected to detect the exact masses of dermorphin and dermorphin (1-4) at m/z 803.37226 and 457.20816, respectively. The SRM method used in the MS/MS confirmation protocol presented high specificity and sensitivity. The selected product ions for dermorphin were 602.2, 202.1 and 574.3 and for dermorphin (1-4) were 207.1, 223.1, and 235.1. Both methods were evaluated for specificity, repeatability, carryover, matrix effects, and recovery. No carryover and matrix effects were detected. The limit of detection for initial testing procedure and the limit of identification for confirmation procedure was 2.5 ng/ml. Also, specificity and robustness were acceptable for the application. Together, the developed methods proved to be efficient for the analysis of dermorphin and metabolite for human doping control purpose.

Detection of ESAs in equine urine and blood by SAR-PAGE.

AbstractErythropoiesis‐stimulating agents (ESAs) have been used in horses for doping purposes to increase the performance of these animals in endurance sports. Currently, enzyme‐linked immunosorbent assay (ELISA) and mass spectrometry methods are used to detect ESA abuse in equines. However, the sarcosyl polyacrylamide gel‐electrophoresis (SAR‐PAGE) technique could also be used, since its application in human doping control is well established and has proven to be more sensitive. In this work, the SAR‐PAGE method was used to detect recombinant human erythropoietin (rHuEPO), novel erythropoiesis stimulating protein (NESP), continuous erythropoietin receptor activator (CERA), and fusion protein of erythropoietin with human immunoglobulin heavy chain Fc region (EPO‐Fc) in horse blood and urine. The purification technique for human blood using MAIIA kits worked well for horse samples. The major challenge was horse urine immunopurification, which proved difficult due to filter clogging, but heating and cooling of the horse urine followed by filtration in 30‐kDa molecular weight cut‐off filters solved this problem. The limits of detection (LODs) of 1.3, 1.6, 6.6, and 13.3 pg/mL for rHuEPO, NESP, CERA, and EPO‐Fc, respectively, obtained in spiked urine and 40, 100, 80, and 400 pg/mL for rHuEPO, NESP, CERA, and EPO‐Fc, respectively, acquired in spiked blood are lower than the LODs reported in the literature using liquid chromatography–mass spectrometry (LC–MS) methods. In addition, the presence of ESAs was detected up to 9 days after the administration of microdoses of Hemax (rHuEPO), NESP, and CERA in horse blood and urine. SAR‐PAGE may be implemented in the routine analysis of horse doping control laboratories for screening and confirmation of ESA abuse, mainly due to its high sensitivity for both matrices compared to published mass spectrometric methods.

Development and validation of an open screening method for doping substances in urine by gas chromatography quadrupole time-of-flight mass spectrometry

In anti-doping, a high number of classes of substances are prohibited and laboratories need to detect these at low urinary concentrations. Traditionally, testing is done using complimentary liquid chromatography mass spectrometry and gas chromatography mass spectrometry. High resolution mass spectrometric acquisition has some important advantages over triple quadrupole instruments (e.g., open screening due to full scan high resolution data acquisition with retrospectivity, compatibility with libraries and a straightforward and effortless addition and validation of new compounds in the future). Doping samples can be stored for 10 years and retrospective data analysis can be used to re-evaluate previously acquired data (e.g., searching for prohibited (designer) substances that were unknown at the initial moment of analysis). During the past decade, these advantages have led to the wide-scale transfer of liquid chromatography triple quadrupole mass spectrometry screening to liquid chromatography high resolution mass spectrometry screening for doping control purposes. Up to now, for gas chromatography a similar switch to high resolution screening has not yet occurred, because so far no method has been developed that combines sufficient sensitivity with wide-scale drug detection.In this work, the current gas chromatography triple quadrupole mass spectrometry screening method for human doping control purposes was successfully converted into an equivalent and complete gas chromatography high resolution acquisition screening method. This new screening method on a gas chromatography quadrupole time-of-flight mass spectrometer has been developed and validated. The method is compliant with the World Anti-Doping Agency requirements and allows the detection of 294 target compounds (and 14 internal standards), including diuretics, stimulants, narcotics, beta-2-agonists, beta-blockers, hormone modulators, anabolic agents and the quantification of 14 endogenous steroids in a single fast run (14.1 min).

Peptide selection for the quantification of P-III-NP in human serum by mass spectrometry.

Procollagen III amino-terminal propeptide (P-III-NP) is currently monitored in human doping control as a biomarker for growth hormone administration and also in clinical diagnostics using immunoassays. Drawbacks to this approach have been highlighted and research is ongoing to develop a mass spectrometric method to complement these methods. However, a lack of traceable reference material, the presence of post-translational modifications (PTMs), and small blood concentration complicate the development of targeted analytical methods for P-III-NP quantification. Tryptic digest products of P-III-NP were assessed by liquid chromatography/mass spectrometry (LC/MS). In silico digestion was used to predict P-III-NP peptides for MS analysis; however, these excluded PTMs. With a priori knowledge of PTMs, we associated experimental P-III-NP peptides with those derived by in silico digestion. Synthesized P-III-NP peptides, hT1 (human) and T5 (human/bovine), were used to develop sensitive micro- and nano-flow LC/MS methods to analyse P-III-NP originating from human serum semi-quantitatively. P-III-NP peptides, T1 and T5, were identified using high-resolution accurate MS (HRAMS). PTMs modified the mass of observed peptides. N-terminal pyroglutamation (pE) in T1 and several hydroxylated prolines (hP) in T5 (G-X-hP motif) were observed. With PTM, hT1 and T5 were observed in a digest of immuno-captured P-III-NP by LC/MS. Using a semi-quantitative approach, hP-III-NP at basal concentrations of 2ng/mL (50pmol) could be estimated from a 200-μL sample volume. Consideration of PTMs is needed to identify P-III-NP peptides produced by digestion with trypsin. The information presented here now gives the most appropriate peptide sequences for synthesizing suitable reference materials required for quantification of human P-III-NP in blood and evidences methodology that is sufficiently sensitive to develop a quantitative method.

Equine in vivo-derived metabolites of the SARM LGD-4033 and comparison with human and fungal metabolites

LGD-4033 has been found in human doping control samples and has the potential for illicit use in racehorses as well. It belongs to the pharmacological class of selective androgen receptor modulators (SARMs) and can stimulate muscle growth, much like anabolic steroids. However, SARMs have shown superior side effect profiles compared to anabolic steroids, which arguably makes them attractive for use by individuals seeking an unfair advantage over their competitors. The purpose of this study was to investigate the metabolites formed from LGD-4033 in the horse in order to find suitable analytical targets for doping controls. LGD-4033 was administered to three horses after which plasma and urine samples were collected and analyzed for metabolites using ultra high performance liquid chromatography coupled to a high resolution mass spectrometer. In horse urine, eight metabolites, both phase I and phase II, were observed most of which had not been described in other metabolic systems. Six of these were also detected in plasma. The parent compound was detected in plasma, but not in non-hydrolyzed urine. The longest detection times were observed for unchanged LGD-4033 in plasma and in urine hydrolyzed with β-glucuronidase and is thus suggested as the analytical target for doping control in the horse. The metabolite profile determined in the horse samples was also compared to those of human urine and fungal incubate from Cunninghamella elegans. The main human metabolite, dihydroxylated LGD-4033, was detected in the horse samples and was also produced by the fungus. However, it was a not a major metabolite for horse and fungus, which highlights the importance of performing metabolism studies in the species of interest.

Determination and confirmation of selective estrogen receptor modulators (SERMs), anti-estrogens and aromatase inhibitors in bovine and porcine urine using UHPLC-MS/MS

ABSTRACTSelective estrogen receptor modulators (SERMs), anti-estrogens and aromatase inhibitors are prohibited in human sports doping. However, they also present a risk of being used illegally in animal husbandry for fattening purposes. A method was developed and validated using UHPLC-MS/MS for the determination and confirmation of SERMs, anti-estrogens and aromatase inhibiters in bovine and porcine urine. This method was used in a survey of more than 200 bovine and porcine urine samples from Dutch farms. In 18 out of 103 porcine urine samples (17%) and two out of 114 bovine samples (2%) formestane, an aromatase inhibitor, was detected. None of the other compounds was detected. From human doping control it is known that formestane can, in some cases, be of natural origin. Analyses of reference samples from untreated bovine and porcine animals demonstrated the presence of formestane in bovine animals, but not yet in porcine animals. Future research will focus on whether the detected formestane in porcine and bovine urine is from endogenous or exogenous origin, using GC-c-IRMS.

Investigation of the metabolites of the HIF stabilizer FG-4592 (roxadustat) in five different in vitro models and in a human doping control sample using high resolution mass spectrometry

FG-4592 is a hypoxia-inducible factor (HIF) stabilizer, which can increase the number of red blood cells in the body. It has not been approved by regulatory authorities, but is available for purchase on the Internet. Due to its ability to improve the oxygen transportation mechanism in the body, FG-4592 is of interest for doping control laboratories, but prior to this study, little information about its metabolism was available. In this study, the metabolism of FG-4592 was investigated in a human doping control sample and in five in vitro models: human hepatocytes and liver microsomes, equine liver microsomes and S9 fraction and the fungus Cunninghamella elegans. By using liquid chromatography coupled to a Q-TOF mass spectrometer operated in MSE and MSMS modes, twelve different metabolites were observed for FG-4592. One monohydroxylated metabolite was detected in both the human and equine liver microsome incubations. For the fungus Cunninghamella elegans eleven different metabolites were observed of which the identical monohydroxylated metabolite had the highest response. This rich metabolic profile and the higher levels of metabolites produced by Cunninghamella elegans demonstrates its usefulness as a metabolite producing medium. In the doping control urine sample, one metabolite, which was the result of a direct glucuronidation, was observed. No metabolites were detected in neither the human hepatocyte nor in the equine liver S9 fraction incubates.

Current status and recent advantages in derivatization procedures in human doping control.

Derivatization is one of the most important steps during sample preparation in doping control analysis. Its main purpose is the enhancement of chromatographic separation and mass spectrometric detection of analytes in the full range of laboratory doping control activities. Its application is shown to broaden the detectable range of compounds, even in LC-MS analysis, where derivatization is not a prerequisite. The impact of derivatization initiates from the stage of the metabolic studies of doping agents up to the discovery of doping markers, by inclusion of the screening and confirmation procedures of prohibited substances in athlete's urine samples. Derivatization renders an unlimited number of opportunities to advanced analyte detection.

Analytical approaches for the detection of emerging therapeutics and non-approved drugs in human doping controls

The number and diversity of potentially performance-enhancing substances is continuously growing, fueled by new pharmaceutical developments but also by the inventiveness and, at the same time, unscrupulousness of black-market (designer) drug producers and providers. In terms of sports drug testing, this situation necessitates reactive as well as proactive research and expansion of the analytical armamentarium to ensure timely, adequate, and comprehensive doping controls. This review summarizes literature published over the past 5 years on new drug entities, discontinued therapeutics, and ‘tailored’ compounds classified as doping agents according to the regulations of the World Anti-Doping Agency, with particular attention to analytical strategies enabling their detection in human blood or urine. Among these compounds, low- and high-molecular mass substances of peptidic (e.g. modified insulin-like growth factor-1, TB-500, hematide/peginesatide, growth hormone releasing peptides, AOD-9604, etc.) and non-peptidic (selective androgen receptor modulators, hypoxia-inducible factor stabilizers, siRNA, S-107 and ARM036/aladorian, etc.) as well as inorganic (cobalt) nature are considered and discussed in terms of specific requirements originating from physicochemical properties, concentration levels, metabolism, and their amenability for chromatographic-mass spectrometric or alternative detection methods.

Identification of sympathomimetic alkylamine agents in urine using liquid chromatography–mass spectrometry and comparison of derivatization methods for confirmation analyses by gas chromatography–mass spectrometry

The detection of 11 sympathomimetic alkylamines in urine was presented with a focus on human doping control is proposed using liquid chromatography tandem mass spectrometry (LC-QqQ) and high resolution mass spectrometry (LC–HRMS) as a screening tool after a dilute-and-shoot (DS) approach. For the LC–HRMS analyses, several compounds exhibited better limits of detection (L.O.D.) than the LC-QqQ. However, due to their small differences in structure, co-elution among the alkylamines was observed. Therefore, the chemical conversion of the alkylamines into an appropriate derivative for the confirmation analyses using gas chromatography–mass spectrometry (GC–MS) was evaluated. Five derivatization approaches were evaluated in an attempt to increase the analytical response and the confidence of the identification. The choice of the appropriated derivative for each alkylamine makes their spectra more easily interpretable, fulfills the WADA's rather strict identification criteria and enables the unequivocal identification of alkylamines in urine.

Recent Developments in Ms For Small Molecules: Application to Human Doping Control Analysis

Recent developments in MS for the detection of small molecules in the context of doping control analysis are reviewed. Doping control analysis is evolving together with MS, which is the technique of choice in order to accomplish the analytical requirements in this field. Since these analytical requirements for the detection of a doping agent depend on the substance, in the first section we review the different scenarios. The commonly established approaches, together with their achievements and drawbacks are described. New developments in hyphenated MS techniques (both GC-MS/MS and LC-MS/MS) concerning interfaces and analyzers are mentioned. The use (or potential use) of these developments in order to minimize the limitations of the commonly established approaches in the doping control field is discussed. Finally, a brief discussion about trends and remaining limitations is presented.

Analysis of octopamine in human doping control samples

The biogenic amine octopamine [4-(2-amino-1-hydroxyethyl)phenol] is prohibited in sports owing to its stimulating and performance-enhancing properties. Adverse analytical findings in athletes' doping control samples commonly result from surreptitious applications; however, the occurrence of octopamine in nutritional supplements and in selected invertebrates as well as the assumption that its N-methylated analog synephrine [4-(1-hydroxyethyl-2-methylamino)phenol, not banned by anti-doping authorities but currently monitored in prevalence studies) might be converted in-vivo into octopamine have necessitated a study to investigate the elimination of synephrine and octopamine present in over-the-counter products. Urine samples collected after administration of nutritional supplements containing octopamine and/or synephrine as well as urine samples collected after therapeutic application of octopamine- or synephrine-containing drugs were analyzed using a validated solid-phase extraction-based procedure employing a weak cation exchange resin and liquid chromatographic/tandem mass spectrometric detection with electrospray ionization and multiple reaction monitoring. In the case of therapeutic octopamine application, the urinary concentration of the target compound increased from baseline levels below the lower limit of detection to 142 µg/mL, while urine samples collected after synephrine as well as dietary supplement administration did not yield any evidence for elevated renal excretion of octopamine.