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Various substances classified by the World Anti-Doping Agency (WADA) as prohibited in sports feature one or more chiral centers. Amongst those, few analytes exist that are so-called threshold substances, for which also enantiomerically pure drugs are available. The commonly employed non-chiral analysis of these compounds does not allow for differentiating between the use of a racemic mixture of e.g. salbutamol and formoterol from their enantiomerically pure analogs. In order to support identifying the exclusive use of the pharmacologically active compound, a multi-analyte chiral chromatography-based analytical approach was developed. The test method considering the β2-agonists fenoterol, formoterol, and salbutamol, the stimulant methamphetamine, the anabolic agent clenbuterol, and the β-blockers metoprolol, pindolol, and propranolol was based on liquid chromatography with a chiral column comprising a stationary phase based on the macrocyclic glycopeptide antibiotic teicoplanin as chiral selector. The liquid chromatograph was interfaced via electrospray ionization to a high resolution/high accuracy mass spectrometer, and urine samples were prepared for analysis following a protocol including enzymatic hydrolysis and subsequent liquid-liquid extraction. For proof-of-concept, authentic urine samples containing the target compounds were analyzed and their enantiomeric composition was assessed. The approach proved suitable for the chiral separation of a total of eight selected enantiomeric doping agents, allowing to determine their ratio at urinary concentrations relevant for sports drug testing purposes, i.e. between 0.01 and 2 ng/mL. Enantioselective assays provide the tool to overcome the limitations of non-chiral approaches in routine doping analysis and can offer support in studies where questions of pharmacokinetics and stereoselectivity are to be addressed for result management and decision-making processes.

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 (pCPA), which is also employed as target analyte in sports drug testing to detect the use of the prohibited nootropic stimulant meclofenoxate. In order 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 post-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 by 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 approximately 1500 ng/mL and 2300 ng/mL after single and multiple sunscreen application, 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 whether chlorphenesin or the prohibited nootropic meclofenoxate was administered.

To date, blood (and serum) as well as urine samples are the most commonly collected specimens for routine doping controls, which allow for the analytical coverage of an extensive set of target analytes relevant to sports drug testing programs. In the course of studies to identify potential alternative matrices to complement current testing approaches, exhaled breath (EB) has been found to offer advantageous properties especially with regard to the sample collection procedure, which is less invasive, less intrusive, and less time‐consuming when compared to conventional blood and urine testing. A yet unaddressed question has been the potential contribution of oral fluid (OF) to EB samples. The current investigation focused on characterizing an electret membrane‐based EB collection device concerning a potential introduction of OF during the sampling procedure. For that purpose, EB and OF samples collected under varying conditions from a total of 14 healthy volunteers were tested for the presence of abundant salivary proteins using bottom‐up proteomics approaches such as SDS‐PAGE followed by tryptic digestion and chromatographic‐mass spectrometric analysis. The trapping baffles integrated into the mouthpiece of the EB collection device were found to effectively retain OF introduced into the unit during sample collection as no saliva breakthrough was detectable using the established analytical approach targeting predominantly the highly abundant salivary α‐amylase. Since α‐amylase was found unaffected by storage, smoking, food intake, and exercise, it appears to be a useful marker to reveal possible OF contaminations of EB collection devices., The investigation focused on characterizing an electret membrane‐based exhaled breath collection device concerning a potential introduction of oral fluid during the sampling procedure by using bottom‐up proteomics approaches. The trapping baffles integrated into the mouthpiece were found to effectively retain oral fluid introduced into the unit during exhaled breath sample collection. Since α‐amylase was found unaffected by storage, smoking, food intake, and exercise, it appears to be a useful marker to reveal possible oral fluid contaminations.

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.

The possibility of nutritional supplement contamination with minute amounts of the selective androgen receptor modulator (SARM) ostarine has become a major concern for athletes and result managing authorities. In case of an adverse analytical finding (AAF), affected athletes need to provide conclusive information, demonstrating that the test result originates from a contamination scenario rather than doping. The aim of this research project was to study the elimination profiles of microdosed ostarine and characterize the time-dependent urinary excretion of the drug and selected metabolites. Single- and multi-dose administration studies with 1, 10, and 50 μg of ostarine were conducted, and collected urine samples were analyzed by LC-MS/MS following solid-phase extraction or enzymatic hydrolysis combined with liquid-liquid extraction. In the post-administration samples, both the maximum urine concentrations/abundance ratios and detection times of ostarine and its phase-I and phase-II metabolites were found to correlate with the administered drug dose. With regard to the observed maximum levels of ostarine, the time points of peak urinary concentrations/abundance ratios, and detection windows, a high inter-individual variation was observed. However, the study demonstrated that a single oral dose of as little as 1 μg can be detected for up to 9 (5) days by monitoring ostarine (glucuronide), and hydroxylated metabolites (especially M1a) appear to offer a considerably shorter detection window. The obtained data on ostarine (metabolite) detection times and urinary concentrations following different administration schemes support the interpretation of AAFs, in particular when scenarios of proven supplement contamination are discussed and supplement administration protocols exist.

Cobaltous ions (Co2+ ) stabilize HIFα, increase endogenous erythropoietin (EPO) production, and may, therefore, be used as a performance-enhancing substance. To date, the dosage necessary to stimulate erythropoiesis is unknown. The aim of this study was, therefore, to determine the minimum dosage necessary to increase erythropoietic processes. In a first double-blind placebo-controlled study (n = 5), single oral Co2+ dosages of 5 mg (n = 6) and 10 mg (n = 7) were administered to healthy young men. Cubital venous blood and urine samples were collected before and up to 24 hours after Co2+ administration. In a second study, the same daily Co2+ dosages were administered for five days (placebo: n = 5, 5 mg: n = 9, 10 mg: n = 7). Blood and urine samples were taken the day before administration and at day 3 and day 5. Plasma [EPO] was elevated by 20.5 ± 16.9% at 5 hours after the single 5-mg administration (p < 0.05) and by 52.8 ± 23.5% up to 7 hours following the 10-mg Co2+ administration (p < 0.001). Urine [Co2+ ] transiently increased, with maximum values 3-5 hours after Co2+ ingestion (5 mg: from 0.8 ± 1.1 to 153.6 ± 109.4 ng/mL, 10 mg: from 1.3 ± 1.7 to 338.0 ± 231,5 ng/mL). During the five days of Co2+ application, 5 mg showed a strong tendency to increase [EPO], while the 10-mg application significantly increased [EPO] at day 5 by 27.2 ± 26.4% (p < 0.05) and the immature reticulocyte fraction by 49.9 ± 21.7% (p < 0.01). [Ferritin] was decreased by 12.4 ± 10.4 ng/mL (p < 0.05). An oral Co2+ dosage of 10 mg/day exerts clear erythropoietic effects, and 5 mg/day tended to increase plasma EPO concentration.

Detecting agents allegedly or evidently promoting growth such as human growth hormone (GH) or growth hormone releasing peptides (GHRP) in doping controls has represented a pressing issue for sports drug testing laboratories. While GH is a recombinant protein with a molecular weight of 22 kDa, the GHRPs are short (3–6 amino acids long) peptides with GH releasing properties. The endogenously produced GH (22 kDa isoform) consists of 191 amino acids and has a monoisotopic molecular mass of 22,124 Da. Within this study, a slightly modified form of GH was discovered consisting of 192 amino acids carrying an additional alanine at the N-terminus, leading to a monoisotopic mass of 22,195 Da. This was confirmed by top-down and bottom-up experiments using liquid chromatography coupled to high resolution/high accuracy mass spectrometry. Additionally, three analogues of GHRPs were identified as Gly-GHRP-6, Gly-GHRP-2 and Gly-Ipamorelin, representing the corresponding GHRP extended by a N-terminal glycine residue. The structure of these peptides was characterised by means of high resolution (tandem) mass spectrometry, and for Gly-Ipamorelin and Gly-GHRP-2 their identity was additionally confirmed by custom synthesis. Further, established in-vitro experiments provided preliminary information considering the potential metabolism after administration.

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.

The utility of hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors as a therapeutic means of treating patients suffering from anaemia has been demonstrated for various clinical settings. However, besides this intended use, HIF stabilizers can be the subject of misuse in amateur and elite sports due to their erythropoietic properties, as recently proven by several cases of adverse analytical findings in doping control testing. Consequently, to allow for adequate and comprehensive test methods, knowledge of the drug candidates' metabolism and analytical options enabling appropriate detection windows in sports drug testing samples (i.e., blood and urine) is essential to doping control laboratories. In the present study, a novel HIF prolyl hydroxylase inhibitor referred to as Roxadustat (FG-4592) and main plasma- and urine-derived metabolites were investigated in the context of routine doping control analytical approaches. Liquid chromatography-mass spectrometry-based test methods were used to study the target analytes' dissociation pathways following electrospray ionization and collision-induced dissociation. Diagnostic precursor-product ion pairs were selected to enable the implementation of the intact drug Roxadustat and selected metabolites into multi-analyte initial testing procedures for plasma and urine specimens. The assays were validated in accordance to guidelines of the World Anti-Doping Agency (WADA) and results demonstrated the suitability (fitness-for-purpose) of the employed analytical methods with detection limits ranging from 0.05 to 1 ng/mL and 1 to 5 ng/mL for urine and plasma, respectively. Subsequently, elimination study plasma and urine samples collected up to 167 h post-administration were analyzed using the validated methods, which suggested the use of different target analytes for blood and urine analyses with FG-4592 and its glucuronide, respectively, for optimal detection windows. Additionally, a light-induced rearrangement product (photoisomer) of Roxadustat resulted in the formation of an additional compound of identical mass. Copyright © 2017 John Wiley & Sons, Ltd.

This retrospective study evaluates the content, the destination and the source of 960 postal items seized by the Swiss customs authorities at the Swiss border between 2013 and 2014. The packages were seized because they contained at least one prohibited doping product as identified by the Swiss law on encouraging sports and physical activity. A total number of 1825 different doping products were confiscated from these parcels, accounting for an average of 1.9 doping products per seized item. In 74% of the cases, where seizures were made, anabolic androgenic steroids, mostly testosterone esters, were discovered. An obvious trading channel for doping products was identified in this study. The seized compounds were predominately manufactured in Asian countries, but sent to Switzerland mostly via South Eastern Europe countries. Due to the unique collaboration between the Swiss customs authorities and the national anti-doping agency, this study uncovered an alarming trend of illegal doping product trafficked to Switzerland.

Rationale The issue of illicit performance enhancement spans human and animal sport in presumably equal measure, with prohibited substances and methods of doping conveying both ways. Due to the proven capability of unbound ionic cobalt (Co2+) to stimulate erythropoiesis in humans, both human and equine anti-doping regulations have listed cobalt as a banned substance, and in particular in horse drug testing, thresholds for cobalt concentrations applying to plasma and urine have been suggested or established. Recent reports about the finding of substantial amounts of undeclared nickel in arguably licit performance- and recovery-supporting products raised the question whether the ionic species of this transition metal (Ni2+), which exhibits similar prolyl hydroxylase inhibiting properties to Co2+, has been considered as a substitute for cobalt in doping regimens. Methods Therefore, a pilot study with 200 routine post-competition doping control horse urine samples collected from animals participating in equestrian, gallop, and trotting in Europe was conducted to provide a first dataset on equine urinary Ni2+ concentrations. All specimens were analyzed by conventional inductively coupled plasma mass spectrometry (ICP-MS) to yield quantitative data for soluble nickel. Results Concentrations ranging from below the assay's limit of quantification (LOQ, 0.5 ng/mL) up to 33.4 ng/mL with a mean value (± standard deviation) of 6.1 (±5.1) ng/mL were determined for the total nickel content. Conclusions In horses, nickel is considered a micronutrient and feed supplements containing nickel are available; hence, follow-up studies are deemed warranted to consolidate potential future threshold levels concerning urine and blood nickel concentrations in horses using larger sets of samples for both matrices and to provide in-depth insights by conducting elimination studies with soluble Ni2+-salt species. Copyright © 2016 John Wiley & Sons, Ltd.

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.

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.

Even without clinical approval, many performance-enhancing drugs are available on the black market and can therefore be easily obtained by cheating athletes. The misuse of these preparations can be associated with unforeseeable health risks - either due to a poor quality of the drugs or as a result of an insufficient clinical assessment. Moreover, confiscated black market products have frequently been shown to contain ingredients other than those declared on the label as well as additional by-products or compounds with a modified molecular structure. This communication describes the identification of an unknown fusion protein observed in several unlabelled black market products obtained from independent sources. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of the confiscated preparations indicated the presence of an 18-kDa fusion protein consisting of the bacterial redox protein thioredoxin-1 (Trx, 12 kDa) and a 6-kDa peptide of unassigned composition. Trx has no relevance as performance enhancing agent but is routinely used as solubility tag for recombinant protein production. Further evaluation of the acquired MS/MS data revealed both an additional His tag and a thrombin cleavage site between the tags and the presumed bioactive peptide. However, thrombin cleavage of the fusion protein and LC-MS/MS analysis of the resulting peptide fragment finally suggested that the unknown protein is only the product of an empty expression vector without the DNA insert of interest. These findings are a further alarming example for the high level of risk that athletes take when misusing drugs obtained from the black market. Copyright © 2014 John Wiley & Sons, Ltd. Language: en

Since first reports on the impact of metals such as manganese and cobalt on erythropoiesis were published in the late 1920s, cobaltous chloride became a viable though not widespread means for the treatment of anaemic conditions. Today, its use is de facto eliminated from clinical practice; however, its (mis)use in human as well as animal sport as an erythropoiesis-stimulating agent has been discussed frequently. In order to assess possible analytical options and to provide relevant information on the prevalence of cobalt use/misuse among athletes, urinary cobalt concentrations were determined by inductively coupled plasma-mass spectrometry (ICP-MS) from four groups of subjects. The cohorts consisted of (1) a reference population with specimens of 100 non-elite athletes (not being part of the doping control system), (2) a total of 96 doping control samples from endurance sport athletes, (3) elimination study urine samples collected from six individuals having ingested cobaltous chloride (500 µg/day) through dietary supplements, and (4) samples from people supplementing vitamin B12 (cobalamin) at 500 µg/day, accounting for approximately 22 µg of cobalt. The obtained results demonstrated that urinary cobalt concentrations of the reference population as well as the group of elite athletes were within normal ranges (0.1–2.2 ng/mL). A modest but significant difference between these two groups was observed (Wilcoxon rank sum test, p 22 ng/mL) up to 33 h. Oral supplementation of 500 µg of cobalamin did not result in urinary cobalt concentrations > 2 ng/mL. Based on these pilot study data it is concluded that measuring the urinary concentration of cobalt can provide information indicating the use of cobaltous chloride by athletes. Additional studies are however required to elucidate further factors potentially influencing urinary cobalt levels. Copyright © 2014 John Wiley & Sons, Ltd.

Quality assurance of pharmaceutical products is of particular importance and thoroughly controlled. Among these, the preparation of human plasma follows strict guidelines from the point of donor selection to product processing. While various precautions particularly concerning antiviral treatment as well as quality assessment are standard procedure, tests for drug residues are rarely, if at all, conducted with fresh frozen plasma products. With the constantly increasing sensitivity and specificity of modern analytical instruments, the detection of trace amounts of therapeutics in plasma is feasible and can be applied to blood products where considered appropriate. To estimate the prevalence of a selection of commonly prescribed and over-the-counter drugs (including diuretics, beta-receptor blocking agents, contraceptives, β2 -agonists, antibiotics, antidepressants, analgesics, opioids, glucocorticosteroids, benzodiazepines, stimulants, and oral anti-diabetics) as well as cannabinoids in human donor plasma, a total of 100 specimens (61 female, 39 male) collected at the German Red Cross Organization in 2012 was subjected to an established analytical approach. The methodology was based on protein precipitation followed by liquid chromatographic-high resolution/high accuracy mass spectrometric analysis. Following initial test results, confirmatory analyses were conducted with respective reference substances employing a conventional liquid chromatography-triple-quadrupole mass spectrometer (LC-MS/MS) apparatus. Out of one hundred samples, five were found to contain diuretics (four hydrochlorothiazide and one torasemide), five contained beta-receptor blocking agents (four bisoprolol and one metoprolol), one was found with residues of pseudoephedrine (stimulant) and one with drosperinone (contraceptive). Overall, 12% of samples yielded detectable amounts of drug residues at concentrations estimated to levels common to individuals under therapeutic treatment. In addition, six aliquots of different lots of commercially available plasma preparations with solvent-detergent processing were tested. Here, no drug residues of the targeted therapeutics were detected.

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.

Products overtly advertised with arguably licit performanceenhancing properties have been the subject of inquests particularly in the context of human sports drug testing for several years [5, 6, 9, 13]. In investigated products, analyses frequently revealed the presence of (undeclared) anabolic agents or stimulants, being intentionally or inadvertently consumed by athletes. Conversely, substances stimulating erythropoiesis (e. g. erythropoietin, hypoxia-inducible factor (HIF) stabilizers) have rarely been reported as ingredients of such products; however, mixtures of proprietary and thus undisclosed content (arguably intended for veterinary use only) have been observed in the personal environment of elite athletes recently. These mixtures, confiscated or test-purchased from Internet-based suppliers by the Center for Preventive Doping Research and different antidoping organizations, claimed blood-building properties and were therefore forwarded to (bio)chemical analyses concerning erythropoietin (EPO) and its derivatives as well as low molecular mass organic and inorganic HIF stabilizers according to established protocols [13].

In the present study, a screening assay was developed comprising 11 prohibited peptides (

Several selective androgen receptor modulators (SARMs) have been synthesized and investigated in humans, rats, and dogs in the past, but no data are yet available concerning the metabolism of SARMs in horses. The aryl-propionamide-derived drug candidates S24 and S4 (andarine) have a strong androgen receptor binding affinity and show distinctive specific cell answers. Although no SARM drug candidate (aiming for testosterone replacement therapy) has completed clinical trials yet, S4 has been illicitly available via the Internet. These facts led to the prohibition of SARMs by the German equestrian federation, and the (mis)use of such compounds would further represent a doping rule violation in horse racing. In this study, the drug candidates S24 and S4 were subjected to in vitro metabolism experiments with equine liver microsomal preparations from a female Quarter Horse to obtain information about potential target analytes in equine doping control analysis. The enzymatically synthesized metabolites were characterized by liquid chromatography–tandem mass spectrometry and –high-resolution/high-accuracy mass spectrometry. All observed S24 and S4 equine metabolites are in agreement with earlier in vitro and in vivo studies in humans and dogs. Nevertheless, the relative percentage of generated equine metabolites (as determined from the analytes’ response in full-scan chromatography–tandem mass spectrometry and –high-resolution/high-accuracy mass spectrometry measurements) differs considerably from the reported profiles. Although the S24 metabolite pattern is comparably balanced concerning glucuronidated and sulfonated conjugates, the major S4 metabolite was found to be the unconjugated dephenylated compound, with a proportion of more than 90%.

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

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 (

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.

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. 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). 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 β4, and a fusion protein of unknown biological activity. 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’.

Even without clinical approval, many performance-enhancing drugs are available on the black market and can therefore be easily obtained by cheating athletes. The misuse of these preparations can be associated with unforeseeable health risks - either due to a poor quality of the drugs or as a result of an insufficient clinical assessment. Moreover, confiscated black market products have frequently been shown to contain ingredients other than those declared on the label as well as additional by-products or compounds with a modified molecular structure. This communication describes the identification of an unknown fusion protein observed in several unlabelled black market products obtained from independent sources. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of the confiscated preparations indicated the presence of an 18-kDa fusion protein consisting of the bacterial redox protein thioredoxin-1 (Trx, 12 kDa) and a 6-kDa peptide of unassigned composition. Trx has no relevance as performance enhancing agent but is routinely used as solubility tag for recombinant protein production. Further evaluation of the acquired MS/MS data revealed both an additional His tag and a thrombin cleavage site between the tags and the presumed bioactive peptide. However, thrombin cleavage of the fusion protein and LC-MS/MS analysis of the resulting peptide fragment finally suggested that the unknown protein is only the product of an empty expression vector without the DNA insert of interest. These findings are a further alarming example for the high level of risk that athletes take when misusing drugs obtained from the black market.

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.

Since first reports on the impact of metals such as manganese and cobalt on erythropoiesis were published in the late 1920s, cobaltous chloride became a viable though not widespread means for the treatment of anaemic conditions. Today, its use is de facto eliminated from clinical practice; however, its (mis)use in human as well as animal sport as an erythropoiesis-stimulating agent has been discussed frequently. In order to assess possible analytical options and to provide relevant information on the prevalence of cobalt use/misuse among athletes, urinary cobalt concentrations were determined by inductively coupled plasma-mass spectrometry (ICP-MS) from four groups of subjects. The cohorts consisted of (1) a reference population with specimens of 100 non-elite athletes (not being part of the doping control system), (2) a total of 96 doping control samples from endurance sport athletes, (3) elimination study urine samples collected from six individuals having ingested cobaltous chloride (500 µg/day) through dietary supplements, and (4) samples from people supplementing vitamin B12 (cobalamin) at 500 µg/day, accounting for approximately 22 µg of cobalt. The obtained results demonstrated that urinary cobalt concentrations of the reference population as well as the group of elite athletes were within normal ranges (0.1-2.2 ng/mL). A modest but significant difference between these two groups was observed (Wilcoxon rank sum test, p 0.01) with the athletes' samples presenting slightly higher urinary cobalt levels. The elimination study urine specimens yielded cobalt concentrations between 40 and 318 ng/mL during the first 6 h post-administration, and levels remained elevated (22 ng/mL) up to 33 h. Oral supplementation of 500 µg of cobalamin did not result in urinary cobalt concentrations 2 ng/mL. Based on these pilot study data it is concluded that measuring the urinary concentration of cobalt can provide information indicating the use of cobaltous chloride by athletes. Additional studies are however required to elucidate further factors potentially influencing urinary cobalt levels.

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.

Quality assurance of pharmaceutical products is of particular importance and thoroughly controlled. Among these, the preparation of human plasma follows strict guidelines from the point of donor selection to product processing. While various precautions particularly concerning antiviral treatment as well as quality assessment are standard procedure, tests for drug residues are rarely, if at all, conducted with fresh frozen plasma products. With the constantly increasing sensitivity and specificity of modern analytical instruments, the detection of trace amounts of therapeutics in plasma is feasible and can be applied to blood products where considered appropriate. To estimate the prevalence of a selection of commonly prescribed and over-the-counter drugs (including diuretics, beta-receptor blocking agents, contraceptives, β2 -agonists, antibiotics, antidepressants, analgesics, opioids, glucocorticosteroids, benzodiazepines, stimulants, and oral anti-diabetics) as well as cannabinoids in human donor plasma, a total of 100 specimens (61 female, 39 male) collected at the German Red Cross Organization in 2012 was subjected to an established analytical approach. The methodology was based on protein precipitation followed by liquid chromatographic-high resolution/high accuracy mass spectrometric analysis. Following initial test results, confirmatory analyses were conducted with respective reference substances employing a conventional liquid chromatography-triple-quadrupole mass spectrometer (LC-MS/MS) apparatus. Out of one hundred samples, five were found to contain diuretics (four hydrochlorothiazide and one torasemide), five contained beta-receptor blocking agents (four bisoprolol and one metoprolol), one was found with residues of pseudoephedrine (stimulant) and one with drosperinone (contraceptive). Overall, 12% of samples yielded detectable amounts of drug residues at concentrations estimated to levels common to individuals under therapeutic treatment. In addition, six aliquots of different lots of commercially available plasma preparations with solvent-detergent processing were tested. Here, no drug residues of the targeted therapeutics were detected.

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.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.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.

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.

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.

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.

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.