Analysis of Potential Gene Doping Preparations for Transgenic DNA in the Context of Sports Drug Testing Programs.

Gene doping has been classified as a prohibited method by the World Anti-Doping Agency (WADA) and the International Olympic Committee (IOC) for over two decades. As gene therapeutic approaches improve and, concomitantly, safety concerns regarding clinical applications decline, apprehensions about their illicit use in elite sports continue to grow. Two products available via Internet-based providers and advertised as EPO-gene- and IGF1-gene-containing materials were analyzed for the presence of potential gene doping agents using a newly developed analytical approach, allowing for the detection of transgenic DNA corresponding to seven potential targets (EPO, FST, GH1, MSTN (Propeptide), IGF1, VEGFA, and VEGFD). Panel detection was based on a 20-plex polymerase chain reaction (PCR) followed by a single base extension (SBE) reaction and subsequent SBE product analyses via matrix-assisted time-of-flight laser desorption/ionization mass spectrometry (MALDI-TOF MS). Extracts of both products were found to contain transgenic EPO-DNA, while transgenic DNA for IGF-1 was not detected. The results were confirmed using SYBR Green qPCR with primer sets directed against EPO and IGF1 cDNA, and the CMV promotor sequence. In this case study, the detection of authentic (whilst low concentrated) transgenes, potentially intended for gene doping practices in readily available products, is reported for the first time.

Identification and Synthesis of Selected In Vitro Generated Metabolites of the Novel Selective Androgen Receptor Modulator (SARM) 2f.

Among anabolic agents, selective androgen receptor modulators (SARMs) represent a new class of potential drugs that can exhibit anabolic effects on muscle and bone with reduced side effects due to a tissue-selective mode of action. Besides possible medical applications, SARMs are used as performance-enhancing agents in sports. Therefore, they are prohibited by the World Anti-Doping Agency (WADA) in and out of competition. Since their inclusion into the WADA Prohibited List in 2008, there has been an increase in not only the number of adverse analytical findings, but also the total number of SARMs, making continuous research into SARMs an ongoing topic in the field of doping controls. 4-((2R,3R)-2-Ethyl-3-hydroxy-5-oxopyrrolidin-1-yl)-2-(trifluoromethyl)benzonitrile (SARM 2f) is a novel SARM candidate and is therefore of particular interest for sports drug testing. This study describes the synthesis of SARM 2f using a multi-step approach, followed by full characterization using liquid chromatography-high-resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance spectroscopy (NMR). To provide the first insights into its biotransformation in humans, SARM 2f was metabolized using human liver microsomes and the microsomal S9 fraction. A total of seven metabolites, including phase I and phase II metabolites, were found, of which three metabolites were chemically synthesized in order to confirm their structure. Those can be employed in testing procedures for routine doping controls, further improving anti-doping efforts.

Probing for the presence of doping agents in exhaled breath using chromatographic/mass spectrometric approaches.

RATIONALE: Exhaled breath (EB) has been demonstrated to be a promising alternative matrix in sports drug testing due to its non-invasive and non-intrusive nature compared with urine and blood collection protocols. In this study, a pilot-test system was employed to create drug-containing aerosols simulating EB in support of the analytical characterization of EB sampling procedures, and the used analytical method was extended to include a broad spectrum of prohibited substances. METHODS: Artificial and authentic EB samples were collected using sampling devices containing an electret filter, and doping agents were detected by means of liquid chromatography and tandem mass spectrometry with unispray ionization. The analytical approach was characterized with regard to specificity, limits of detection, carry-over, recovery and matrix effects, and the potential applicability to routine doping controls was shown using authentic EB samples collected after single oral dose applications of glucocorticoids and stimulants. RESULTS: The analytical method was found to be specific for a total of 49 model substances relevant in sports drug testing, with detection limits ranging from 1 to 500 pg per cartridge. Both ion suppression (-62%) and ion enhancement (+301%) effects were observed, and all model compounds applied to EB sampling devices were still detected after 28 days of storage at room temperature. Authentic EB samples collected after the oral administration of 10 mg of prednisolone resulted in prednisolone findings in specimens obtained from 3 out of 6 participants up to 2 h. In octodrine, dimethylamylamine (DMAA) and isopropylnorsynephrine post-administration EB samples, the drugs were detected over a period of 50, 48, and 8 h, respectively. CONCLUSIONS: With the analytical approach developed within this study, the identification of a broad spectrum of prohibited doping agents in EB samples was accomplished. Application studies and stability tests provided information to characterize EB as a potential matrix in sports drug testing.

Chromatographic-mass spectrometric analysis of the urinary metabolite profile of chlorphenesin observed after dermal application of chlorphenesin-containing sunscreen.

RATIONALE: Chlorphenesin is an approved biocide frequently used in cosmetics, and its carbamate ester is an approved skeletal muscle relaxant in certain countries for the treatment of discomfort related to skeletal muscle trauma and inflammation. A major urinary metabolite is 4-chlorophenoxy acetic acid (4-CPA), also known as para-chlorophenoxyacetate, which is also employed as a target analyte in sports drug testing to detect the use of the prohibited nootropic stimulant meclofenoxate. To distinguish between 4-CPA resulting from chlorphenesin, chlorphenesin carbamate, and meclofenoxate, urinary metabolite profiles of chlorphenesin after legitimate use were investigated. METHODS: Human administration studies with commercially available sunscreen containing 0.25% by weight of chlorphenesin were conducted. Six study participants dermally applied 8 g of sunscreen and collected urine samples before and up to 7 days after application. Another set of six study participants applied 8 g of sunscreen on three consecutive days, and urine samples were also taken for up to 5 days after the last dosing. Urine specimens were analyzed using liquid chromatography-high resolution (tandem) mass spectrometry, and urinary metabolites were identified in accordance with literature data by accurate mass analysis of respective precursor and characteristic product ions. RESULTS: In accordance with literature data, chlorphenesin yielded the characteristic urinary metabolites, chlorphenesin glucuronide, chlorphenesin sulfate, and 3-(4-chlorophenoxy)-2-hydroxypropanoic acid (4-CPP), as well as the common metabolite 4-CPA. 4-CPA and 4-CPP were observed at similar abundances, with urinary concentrations of 4-CPA reaching up to ~1500 and 2300 ng/mL after single and multiple sunscreen applications, respectively. CONCLUSION: 4-CPA is a common metabolite of meclofenoxate, chlorphenesin, and chlorphenesin carbamate. Monitoring the diagnostic urinary metabolites of chlorphenesin provides conclusive supporting evidence of whether chlorphenesin or the prohibited nootropic meclofenoxate was administered.

Analytical challenges in sports drug testing.

Analytical chemistry represents a central aspect of doping controls. Routine sports drug testing approaches are primarily designed to address the question whether a prohibited substance is present in a doping control sample and whether prohibited methods (for example, blood transfusion or sample manipulation) have been conducted by an athlete. As some athletes have availed themselves of the substantial breadth of research and development in the pharmaceutical arena, proactive and preventive measures are required such as the early implementation of new drug candidates and corresponding metabolites into routine doping control assays, even though these drug candidates are to date not approved for human use. Beyond this, analytical data are also cornerstones of investigations into atypical or adverse analytical findings, where the overall picture provides ample reason for follow-up studies. Such studies have been of most diverse nature, and tailored approaches have been required to probe hypotheses and scenarios reported by the involved parties concerning the plausibility and consistency of statements and (analytical) facts. In order to outline the variety of challenges that doping control laboratories are facing besides providing optimal detection capabilities and analytical comprehensiveness, selected case vignettes involving the follow-up of unconventional adverse analytical findings, urine sample manipulation, drug/food contamination issues, and unexpected biotransformation reactions are thematized.

Expanding analytical options in sports drug testing: Mass spectrometric detection of prohibited substances in exhaled breath.

RATIONALE: Continuously refining and advancing the strategies and methods employed in sports drug testing is critical for efficient doping controls. Besides improving and expanding the spectrum of target analytes, alternative test matrices have warranted in-depth evaluation as they commonly allow for minimal-/non-invasive and non-intrusive sample collection. In this study, the potential of exhaled breath (EB) as doping control specimen was assessed. METHODS: EB collection devices employing a non-woven electret-based air filter unit were used to generate test specimens, simulating a potential future application in doping controls. A multi-analyte sports drug testing approach configured for a subset of 12 model compounds that represent specific classes of substances prohibited in sports (anabolic agents, hormone and metabolic modulators, stimulants, and beta-blockers) was established using unispray liquid chromatography/tandem mass spectrometry (LC/MS/MS) and applied to spiked and elimination study EB samples. The test method was characterized concerning specificity, assay imprecision, and limits of detection. RESULTS: The EB collection device allowed for retaining and extracting all selected model compounds from the EB aerosol. Following elution and concentration, LC/MS/MS analysis enabled detection limits between 5 and 100 pg/filter and imprecisions ranging from 3% to 20% for the 12 selected model compounds. By means of EB samples from patients and participants of administration studies, the elimination of relevant compounds and, thus, their traceability in EB for doping control purposes, was investigated. Besides stimulants such as methylhexaneamine and pseudoephedrine, also the anabolic-androgenic steroid dehydrochloromethyltestosterone, the metabolic modulator meldonium, and the beta-blocker bisoprolol was detected in exhaled breath. CONCLUSIONS: The EB aerosol has provided a promising proof-of-concept suggesting the expansion of this testing strategy as a complement to currently utilized sports drug testing programs.

Qualitative and Semiquantitative Analysis of Doping Products Seized at the Swiss Border.

BACKGROUND: Substances developed for therapeutic use are also known to be misused by athletes as doping agents and, outside of regulated sport, for image-enhancement. This has generated a market for counterfeit doping substances. Counterfeit doping agents may be of poor pharmaceutical quality and therefore constitute health risks to consumers. OBJECTIVES: This study aims to investigate the pharmaceutical quality of 1,190 doping products seized at the Swiss border. METHODS: Swiss customs authorities seize incoming shipments potentially containing doping agents. Qualitative and semiquantitative analyses were performed in order to test for prohibited doping substances. The main analytical methods utilized for characterizing confiscated compounds were liquid chromatography-high resolution mass spectrometry, polyacrylamide gel electrophoresis with subsequent in-gel tryptic digestion and identification of peptidic compounds using nanoliquid chromatography-tandem mass spectrometry, and electrochemiluminescence immuno assay. RESULTS: For 889 (75%) of the analyzed products, the label suggested the content of anabolic agents, for 146 samples (12%) peptide hormones or growth factors, and for 113 items (9%) antiestrogens, aromatase inhibitors or other metabolic modulators. For the majority of the investigated products, the pharmaceutical quality was an unsatisfactory standard: nonapproved substances were detected and less than 20% of the products contained the claimed substance in the respective amount. CONCLUSION: A comprehensive sample of confiscated doping products was analyzed, allowing for monitoring of developments regarding the use of doping substances in Switzerland and for anticipating future trends and challenges in sports drug testing. An alarming number of tested products was of substandard pharmaceutical quality.

Identification of selected in vitro generated phase-I metabolites of the steroidal selective androgen receptor modulator MK-0773 for doping control purposes.

Research into developing anabolic agents for various therapeutic purposes has been pursued for decades. As the clinical utility of anabolic-androgenic steroids has been found to be limited because of their lack of tissue selectivity and associated off-target effects, alternative drug entities have been designed and are commonly referred to as selective androgen receptor modulators (SARMs). While most of these SARMs are of nonsteroidal structure, the drug candidate MK-0773 comprises a 4-aza-steroidal nucleus. Besides the intended therapeutic use, SARMs have been found to be illicitly distributed and misused as doping agents in sport, necessitating frequently updated doping control analytical assays. As steroidal compounds reportedly undergo considerable metabolic transformations, the phase-I metabolism of MK-0773 was simulated using human liver microsomal (HLM) preparations and electrochemical conversion. Subsequently, major metabolic products were identified and characterized employing liquid chromatography-high-resolution/high- accuracy tandem mass spectrometry with electrospray (ESI) and atmospheric pressure chemical ionization (APCI) as well as nuclear magnetic resonance (NMR) spectroscopy. MK-0773 produced numerous phase-I metabolites under the chosen in vitro incubation reactions, mostly resulting from mono- and bisoxygenation of the steroid. HLM yielded at least 10 monooxygenated species, while electrochemistry-based experiments resulted predominantly in three monohydroxylated metabolites. Elemental composition data and product ion mass spectra were generated for these analytes, ESI/APCI measurements corroborated the formation of at least two N-oxygenated metabolites, and NMR data obtained from electrochemistry-derived products supported structures suggested for three monohydroxylated compounds. Hereby, the hydroxylation of the A-ring located N- bound methyl group was found to be of particular intensity. In the absence of controlled elimination studies, the produced information enables the implementation of new target analytes into routine doping controls and expands the focus of anti-doping efforts concerning this new anabolic agent.

In vitro metabolism studies on the selective androgen receptor modulator (SARM) LG121071 and its implementation into human doping controls using liquid chromatography-mass spectrometry.

LG121071 is a member of the tetrahydroquinolinone-based class of selective androgen receptor modulator (SARM) drug candidates. These nonsteroidal compounds are supposed to act as full anabolic agents with reduced androgenic properties. As SARMs provide an alternative to anabolic androgenic steroids, they represent an emerging class of potential doping substances abused by athletes for illicit performance enhancement. According to the World Anti-Doping Agency's regulations, SARMs are banned substances and part of the Prohibited List since 2008. In consideration of the increasing number of adverse analytical findings in doping controls caused by SARMs abuse, potential drug candidates such as LG121071 have been proactively investigated to enable a timely integration into routine testing procedures even though clinical trials are not yet complete. In the present approach, the collision-induced dissociation (CID) of LG121071 was characterized by means of electrospray ionization-high resolution/high accuracy mass spectrometry, MS(n), and isotope labeling experiments. Interestingly, the even-electron precursor ion [M + H](+) at m/z 297 was found to produce a radical cation at m/z 268 under CID conditions, violating the even-electron rule that commonly applies. For doping control purposes, metabolites were generated in vitro and a detection method for urine samples based on liquid chromatography-tandem mass spectrometry was established. The overall metabolic conversion of LG121071 was modest, yielding primarily mono-, bis- and trishydroxylated species. Notable, however, was the identification of a glucuronic acid conjugate of the intact drug, attributed to an N-glucuronide structure. The sample preparation procedure included the enzymatic hydrolysis of glucuronides prior to liquid-liquid extraction, allowing intact LG121071 to be measured, as well as the corresponding phase-I metabolites. The method was characterized concerning inter alia lower limit of detection (0.5 ng mL(-1) in urine), recovery (40%), and intra-/interday precision (2.3% to 11.7%) to assess its fitness for purpose. Prospectively, the assay can serve as detection method for LG121071 in drug testing and/or doping controls.

Identification of black market products and potential doping agents in Germany 2010-2013.

PURPOSE: The desire to increase the athletic performance, to 'optimize' an individual's appearance, and to complement but also to arguably substitute exercise by means of drugs and drug candidates has generated a considerable (illicit) market for compounds such as anabolic-androgenic steroids, stimulants, growth promoting peptide hormones, and so on. Genuinely developed for therapeutic use, their abuse/misuse generates enormous health risks, which has necessitated comprehensive controls of compound trafficking by customs and anti-doping authorities. METHODS: From 2012 to 2013, the Bureau of Customs Investigation confiscated products containing anabolic-androgenic steroids (AAS; 259 kg), stimulants (13 kg), selective estrogen receptor modulators (SERMs; 24 kg), and human growth hormone (hGH; 3500 ampules). In cooperation with the Bureau and under the umbrella of the European Monitoring Center for Emerging Doping Agents (EuMoCEDA), the Cologne Anti-Doping Laboratory analyzed an additional 337 (black market) products between 2010 and 2013, allowing to monitor developments in drug use and, hence, the anticipation of new challenges in sports drug testing. Main tools utilized in characterizing confiscated materials were liquid chromatography-high resolution mass spectrometry (LC-HRMS), gas chromatography-high resolution mass spectrometry (GC-HRMS), and polyacrylamide gel electrophoresis (PAGE) with subsequent bottom-up identification of peptidic compounds using nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS). RESULTS: Among the 337 substances analyzed in the doping control laboratory in Cologne, 67 active ingredients were found, 49 of which being categorized as doping agents by the World Anti-Doping Agency (WADA). A total of 83.7 % accounted for steroidal substances (predominantly testosterone, trenbolone, and nandrolone and corresponding esters), 12.8 % accounted for peptide hormones and growth factors (predominantly hGH and growth hormone releasing peptides (GHRPs)), 3.2 % of the products contained hormones and metabolic modulators, and 0.3 % accounted for diuretic agents. Outstanding findings were the detection of the selective androgen receptor modulator (SARM) LGD-4033, the thymic hormone thymosin ß4, and a fusion protein of unknown biological activity. CONCLUSIONS: Trafficking of considerable amounts of arguably performance and/or body-enhancing compounds has been observed during the past 4 years, the majority of which is categorized as relevant to sports drug testing. Several substances are of fake/non-approved nature and represent enormous health risks to the 'customer'.

Liquid chromatography-high resolution/ high accuracy (tandem) mass spectrometry-based identification of in vivo generated metabolites of the selective androgen receptor modulator ACP-105 for doping control purposes.

Selective androgen receptor modulators (SARMs) represent an emerging class of therapeutics which have been prohibited in sport as anabolic agents according to the regulations of the World Anti-Doping Agency (WADA) since 2008. Within the past three years, numerous adverse analytical findings with SARMs in routine doping control samples have been reported despite missing clinical approval of these substances. Hence, preventive doping research concerning the metabolism and elimination of new therapeutic entities of the class of SARMs are vital for efficient and timely sports drug testing programs as banned compounds are most efficiently screened when viable targets (for example, characteristic metabolites) are identified. In the present study, the metabolism of ACP-105, a novel SARM drug candidate, was studied in vivo in rats. Following oral administration, urine samples were collected over a period of seven days and analyzed for metabolic products by Liquid chromatography-high resolution/high accuracy (tandem) mass spectrometry. Samples were subjected to enzymatic hydrolysis prior to liquid-liquid extraction and a total of seven major phase-I metabolites were detected, three of which were attributed to monohydroxylated and four to bishydroxylated ACP-105. The hydroxylation sites were assigned by means of diagnostic product ions and respective dissociation pathways of the analytes following positive or negative ionization and collisional activation as well as selective chemical derivatization. The identified metabolites were used as target compounds to investigate their traceability in a rat elimination urine samples study and monohydroxylated and bishydroxylated species were detectable for up to four and six days post-administration, respectively.

Monitoring phosphodiesterase-4 inhibitors using liquid chromatography/(tandem) mass spectrometry in sports drug testing.

RATIONALE: The recent discovery of resveratrol's capability to inhibit cAMP-specific phosphodiesterases (PDEs) and, as a consequence, to enhance particularly the activity of Sirt1 in animal models has reinforced the interest of preventive doping research organizations, especially in PDE4 inhibitors. Among these, the archetypical PDE4-inhibitor rolipram significantly increased the number of mitochondria in laboratory rodents, which further demonstrated a performance increase in a treadmill-test (time-to-exhaustion) of approximately 40%. Besides rolipram, a variety of new PDE4-inhibiting substances including cilomilast, roflumilast, and numerous additional new drug entities were described, with roflumilast being the first-in-class having received clinical approval for the treatment of chronic obstructive pulmonary disease (COPD). Due to the availability of these substances, and the fact that a misuse of such compounds in sport cannot be excluded, it deems relevant to probe for the prevalence of these compounds in sports drug testing programs. METHODS: Known urinary phase-I metabolites of rolipram, roflumilast, and cilomilast were generated by in vitro incubations employing human liver microsomal preparations. The metabolites obtained were studied by liquid chromatography with high-resolution/high-accuracy tandem mass spectrometry (LC/MS/MS) and the reference product ion mass spectra of established and most relevant metabolites were utilized to provide the information necessary for comprehensive doping controls. The analytical procedure was based on conventional routine doping control assays employing enzymatic hydrolysis followed by liquid-liquid extraction and subsequent LC/MS/MS measurement. RESULTS: Structures of diagnostic product ions and dissociation pathways of target analytes were elucidated, providing the information required for implementation into an existing test method for routine sports drug testing. The established method allowed for detection limits for the intact drugs of 1-5 ng/mL, and further assay characteristics (intraday precision 1.5-13.7%, interday precision 7.3-18.6%, recovery 20-100%, ion suppression/enhancement, and specificity) were determined. In addition, proof-of-concept analyses concerning roflumilast were conducted with a urine sample obtained from a COPD patient under roflumilast treatment.

Quantification of AICAR-ribotide concentrations in red blood cells by means of LC-MS/MS.

AICAR (5-amino-4-imidazolecarboxyamide ribonucleoside) arguably provides performance-enhancing properties even in the absence of physical exercise and, therefore, the substance is banned in elite sports since 2009. Due to the natural presence of AICAR in human blood and urine, uncovering the misuse by direct qualitative analysis is not possible. Entering the circulation, the riboside is immediately incorporated into red blood cells (RBCs) and transformed into the corresponding ribotide (5'-monophosphate) form. Within the present study, an analytical method was developed to determine AICAR-ribotide concentrations in RBC concentrates by means of liquid chromatography-tandem mass spectrometry. The method was validated enabling quantitative result interpretation considering the parameters specificity, precision (intra- and interday), linearity, recovery, accuracy (LOD/LOQ), stability and ion suppression. By analysing 99 RBC samples of young athletes, normal physiological levels of AICAR-ribotide were determined (10-500 ng/mL), and individual levels were found to be stable for several days. Employing in vitro incubation experiments with AICAR riboside in fresh whole blood samples, the ribotide concentrations were observed to increase significantly within 30 min from baseline to 1-10 µg/mL. These levels are considered conserved for the lifetime of the erythrocyte and, thus, the results of the in vitro model strongly support the hypothesis that measuring abnormally high AICAR-ribotide concentrations in RBC of elite athletes has the potential to uncover the misuse of this substance for a long period of time.

The fungus Cunninghamella elegans can produce human and equine metabolites of selective androgen receptor modulators (SARMs).

1. Selective androgen receptor modulators (SARMs) are a group of substances that have potential to be used as doping agents in sports. Being a relatively new group not available on the open market means that no reference materials are commercially available for the main metabolites. In the presented study, the in vitro metabolism of SARMs by the fungus Cunninghamella elegans has been investigated with the purpose of finding out if it can produce relevant human and equine metabolites. 2. Three different SARMs, S1, S4 and S24, were incubated for 5 days with C. elegans. The samples were analysed both with and without sample pretreatment using ultra performance liquid chromatography coupled to high resolution mass spectrometry. 3. All the important phase I and some phase II metabolites from human and horse were formed by the fungus. They were formed through reactions such as hydroxylation, deacetylation, O-dephenylation, nitro-reduction, acetylation and sulfonation. 4. The study showed that the fungus produced relevant metabolites of the SARMs and thus can be used to mimic mammalian metabolism. Furthermore, it has the potential to be used for future production of reference material.

Determination of urinary deanol-N-oxide excretion profiles after ingestion of nutritional supplements containing deanol.

2-(Dimethylamino)ethan-1-ol (Deanol) is a widely produced chemical used by both industry and consumers in a variety of applications. Meclofenoxate, a stimulant classified on the World Anti-Doping Agency Prohibited List, metabolizes into deanol and, presumably, its main metabolite deanol-N-oxide. Hence, using liquid chromatography-tandem mass spectrometry, a quantitative detection method for deanol-N-oxide in urine was developed. Subsequently, the urinary excretion of deanol-N-oxide after oral application of 130 mg of deanol was determined in six volunteers, and urine samples of a cohort of 180 male and female athletes from different sports were analyzed. In addition, urinary deanol-N-oxide was determined in an exploratory study with one volunteer ingesting 250 mg of meclofenoxate. The developed test method allowed for limits of detection and quantification for deanol-N-oxide at 0.05 and 0.15 µg/mL, respectively. Urinary deanol-N-oxide cmax levels were found between 100 and 250 µg/mL 2-5 h post-administration of 130 mg of deanol. Similarly, urine samples collected after the administration of 250 mg of meclofenoxate exhibited cmax levels of 115 µg/mL. In contrast, deanol-N-oxide urine concentrations of pre-administration specimens and 180 routine doping control urine sample were between 0.3 and 1.3 µg/mL and below limit of quantification and 1.8 µg/mL, respectively. The study suggests that the use of deanol and meclofenoxate results in significantly elevated urinary deanol-N-oxide levels. Whether or not monitoring deanol-N-oxide in doping controls can support decision-making processes concerning the detection of meclofenoxate use necessitates further investigations taking into consideration the elimination kinetics of 4-chlorophenoxyacetic acid, the main metabolite of meclofenoxate, and deanol-N-oxide.

Investigations into Annona fruit consumption as a potential source of dietary higenamine intake in the context of sports drug testing.

Higenamine is prohibited in sports as a ß2 -agonist by the World Anti-Doping Agency. As a key component of a great variety of plants, including the Annonaceae family, one aim of this research project was to evaluate whether the ingestion of Annona fruit could lead to higenamine adverse analytical findings. Single-dose administration studies including three Annona species (i.e., Annona muricata, Annona cherimola, and Annona squamosa) were conducted, leading to higenamine findings below the established minimum reporting level (MRL) of 10 ng/mL in urine. In consideration of cmax values (7.8 ng/mL) observed for higenamine up to 24 h, a multidose administration study was also conducted, indicating cumulative effects, which can increase the risk of exceeding the applicable MRL doping after Annona fruit ingestion. In this study, however, the MRL was not exceeded at any time point. Further, the major urinary excretion of higenamine in its sulfo-conjugated form was corroborated, its stability in urine was assessed, and in the absence of reference material, higenamine sulfo-conjugates were synthesized and comprehensively characterized, suggesting the predominant presence of higenamine 7-sulfate. In addition, the option to include complementary biomarkers of diet-related higenamine intake into routine doping controls was investigated. A characteristic urinary pattern attributed to isococlaurine, reticuline, and a yet not fully characterized bismethylated higenamine glucuronide was observed after Annona ingestion but not after supplement use, providing a promising dataset of urinary biomarkers, which supports the discrimination between different sources of urinary higenamine detected in sports drug testing programs.

Metabolism of growth hormone releasing peptides.

New, potentially performance enhancing compounds have frequently been introduced to licit and illicit markets and rapidly distributed via worldwide operating Internet platforms. Developing fast analytical strategies to follow these new trends is one the most challenging issues for modern doping control analysis. Even if reference compounds for the active drugs are readily obtained, their unknown metabolism complicates effective testing strategies. Recently, a new class of small C-terminally amidated peptides comprising four to seven amino acid residues received considerable attention of sports drug testing authorities due to their ability to stimulate growth hormone release from the pituitary. The most promising candidates are the growth hormone releasing peptide (GHRP)-1, -2, -4, -5, -6, hexarelin, alexamorelin, and ipamorelin. With the exemption of GHRP-2, the entity of these peptides represents nonapproved pharmaceuticals; however, via Internet providers, all compounds are readily available. To date, only limited information on the metabolism of these substances is available and merely one metabolite for GHRP-2 is established. Therefore, a comprehensive in vivo (po and iv administration in rats) and in vitro (with human serum and recombinant amidase) study was performed in order to generate information on urinary metabolites potentially useful for routine doping controls. The urine samples from the in vivo experiments were purified by mixed-mode cation-exchange solid-phase extraction and analyzed by ultrahigh-performance liquid chromatography (UHPLC) separation followed by high-resolution/high-accuracy mass spectrometry. Combining the high resolution power of a benchtop Orbitrap mass analyzer for the first metabolite screening and the speed of a quadrupole/time-of-flight (Q-TOF) instrument for identification, urinary metabolites were screened by means of a sensitive full scan analysis and subsequently confirmed by high-accuracy product ion scan experiments. Two deuterium-labeled internal standards (triply deuterated GHRP-4 and GHRP-2 metabolite) were used to optimize the extraction and analysis procedure. Overall, 28 metabolites (at least three for each GHRP) were identified from the in vivo samples and main metabolites were confirmed by the human in vitro model. All identified metabolites were formed due to exopeptidase- (amino- or carboxy-), amidase-, or endopeptidase activity.

Mass spectrometric identification and characterization of urinary metabolites of isopropylnorsynephrine for doping control purposes.

Isopropylnorsynephrine (isopropyloctopamine, deterenol, 4-(1-hydroxy-2-(isopropylamino)ethyl)phenol), a beta-selective and direct-acting adrenergic agonist, has been reported in the past as declared as well as non-declared ingredient of dietary supplements. The proven biological activity and the structural similarity of isopropylnorsynephrine to substances classified as prohibited compounds according to the World Anti-Doping Agency's (WADA's) regulations could necessitate the inclusion of this sympathomimetic amine into routine doping control analytical assays. Therefore, information on urinary metabolites is desirable in order to allow for an efficient implementation of target compounds into existing multi-analyte testing procedures, enabling the unequivocal identification of the administration of isopropylnorsynephrine by an athlete. In a pilot study setting, urine samples were collected prior to and after the oral application of ca. 8.7 mg of isopropylnorsynephrine, which were subjected to liquid chromatography-high resolution/high accuracy (tandem) mass spectrometry. The intact drug, hydroxylated and/or glucurono- or sulfo-conjugated isopropylnorsynephrine were detected up to 48 h post-administration, with isopropylnorsynephrine sulfate representing the most abundant urinary target analyte. No relevant amounts of the dealkylation product (octopamine) were observed, indicating that merely moderate adaptations of existing test methods (or data evaluation strategies) are required to include isporpoylnorsynephrine in antidoping analytics, if required.

Comprehensive plasma-screening for known and unknown substances in doping controls.

Occasionally, doping analysis has been recognized as a competitive challenge between cheating sportsmen and the analytical capabilities of testing laboratories. Both have made immense progress during the last decades, but obviously the athletes have the questionable benefit of frequently being able to switch to new, unknown and untested compounds to enhance their performance. Thus, as analytical counteraction and for effective drug testing, a complementary approach to classical targeted methods is required in order to implement a comprehensive screening procedure for known and unknown xenobiotics. The present study provides a new analytical strategy to circumvent the targeted character of classical doping controls without losing the required sensitivity and specificity. Using 50 microL of plasma only, the method potentially identifies illicit drugs in low ng/mL concentrations. Plasma provides the biological fluid with the circulating, unmodified xenobiotics; thus the identification of unknown compounds is facilitated. After a simple protein precipitation, liquid chromatographic separation and subsequent detection by means of high resolution/high accuracy orbitrap mass spectrometry, the procedure enables the determination of numerous compounds from different classes prohibited by the World Anti-Doping Agency (WADA). A new hyphenated mass spectrometry technology was employed without precursor ion selection for higher collision energy dissociation (HCD) fragmentation experiments. Thus the mass spectra contained all the desired information to identify unknown substances retrospectively. The method was validated for 32 selected model compounds for qualitative purposes considering the parameters specificity, selectivity, limit of detection (<0.1-10 ng/mL), precision (9-28%), robustness, linearity, ion suppression and recovery (80-112%). In addition to the identification of unknown compounds, the plasma samples were simultaneously screened for known prohibited targets.

Quantification of urinary AICAR concentrations as a matter of doping controls.

Influencing the endurance in elite sports is one of the key points in modern sports science. Recently, a new class of prohibited substances reached in the focus of doping control laboratories and their misuse was classified as gene doping. The adenosine monophosphate activated protein kinase activator 5-amino-4-imidazolecarboxyamide ribonucleoside (AICAR) was found to significantly enhance the endurance even in sedentary mice after treatment. Due to endogenous production of AICAR in healthy humans, considerable amounts were present in the circulation and, thus, were excreted into urine. Considering these facts, the present study was initiated to fix reference values of renally cleared AICAR in elite athletes. Therefore a quantitative analytical method by means of isotope-dilution liquid chromatography (analytical column: C6-phenyl) coupled to tandem mass spectrometry, after a sample preparation consisting of a gentle dilution of native urine, was developed. Doping control samples of 499 athletes were analysed, and AICAR concentrations in urine were determined. The mean AICAR value for all samples was 2,186 ng/mL with a standard deviation of 1,655 ng/mL. Concentrations were found to differ depending on gender, type of sport and type of sample collection (in competition/out of competition). The method was fully validated for quantitative purposes considering the parameters linearity, inter- (12%, 7% and 10%) and intraday precision (14%, 9% and 12%) at low, mid and high concentration, robustness, accuracy (approx. 100%), limit of quantification (100 ng/mL), stability and ion suppression effects, employing an in-house synthesised (13)C(5)-labelled AICAR as internal standard.