Elimination profile of low-dose chlortalidone and its detection in hair for doping analysis-Implication for unintentional non-therapeutic exposure.

Chlortalidone (CLT) is a thiazide-type diuretic with high affinity for the erythrocyte carbonic anhydrase. Therapeutically, it is mostly used to treat edema and hypertension due to liver cirrhosis, heart insufficiency, or renal dysfunction. Although diuretics and masking agents are prohibited by the World Anti-Doping Agency (WADA) at all times in sports, substances belonging to this category are constantly detected in athlete samples, according to WADA's annual testing figures. Within this group of structurally diverse compounds, a threshold of 20 ng/mL has been introduced for six substances solely due to their presence as contaminants in other permitted drugs because of pharmaceutical production processes. In a recent presumptive doping case with a low urinary CLT concentration, the question of unintentional doping, for example, by contaminated non-steroidal anti-inflammatory drug intake, arose. To examine this potential scenario, a co-elimination of low-dose CLT and hydrochlorothiazide (HCTA; 20 × 50 µg, 0.2 mg/day each) was conducted on five consecutive days in two volunteers. Urine samples were subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS). Moreover, we examined the incorporation of CLT in scalp hair. HCTA is rapidly excreted renally in comparatively high concentrations. In contrast, the elimination of CLT is considerably slower (terminal elimination half-life extended by a factor of 12) and, consequently, much less concentrated in corresponding urine samples (45 and 53 ng/mL, respectively). Conversely, a higher hair incorporation of chlorthalidone was observed with simultaneous dosing of both. The results suggest that an unintentional intake of sub-therapeutic CLT doses due to contamination might result in an adverse analytical finding.

Detection of 18-methyl steroids: Case report on a forensic urine sample and corresponding dietary supplements.

The detection of a putative 18-methyl-19-nortestosterone metabolite in a forensic bodybuilder's urine sample collected as part of a criminal proceeding has triggered a follow-up investigation. Four different dietary supplements in the possession of the suspect were examined with regard to possible precursor steroids. This led to the detection of the declared ingredient methoxydienone, which was confirmed by both, GC-MSMS and LC-HRMSMS. As neither 18-methyl-testosterone, nor 18-methyl-19-nortestosterone were detectable in the supplements, the possibility that the metabolite originates from methoxydienone was investigated. For this purpose, the metabolic fate of methoxydienone was studied in vitro using human HepG2 cells and in vivo by a single oral administration. While the 18-methyl-19-nortestosterone metabolite was not generated by HepG2 cells incubated with methoxydienone, it was observed in the urine samples collected at 2, 6, 10 and 24 h after methoxydienone administration. Moreover, the potential binding of methoxydienone as ligand to the human androgen receptor was modelled in silico in comparison with 18-methylnandrolone, for which androgen receptor activation had been shown in an in vitro approach before. In conclusion, we could ascribe the presence of the 18-methyl-19-nortestosterone metabolite in a forensic urine sample to originate from methoxydienone present in dietary supplements. Methoxydienone was observed to slowly degrade by demethylation of the methoxy substituent in liquid solutions. While no compound-specific intermediates were identified that allowed differentiation from other 18-methyl steroids, the 18-methyl-19-nortestosterone metabolite proved to be a suitable marker for reliable detection in doping analysis.

Detection of the selective androgen receptor modulator GSK2881078 and metabolites in urine and hair after single oral administration.

Hair and urine concentrations of the nonsteroidal selective androgen receptor modulator GSK2881078 were examined following single oral administration to investigate its hair incorporation and estimate the general suitability of hair testing for selected androgen receptor modulators. Hair segments were collected following a single dose of 1.5 mg GSK2881078 by repeated shaving of scalp hair at Week 0 (blank), Week 1 (representing the pre-application period), Week 3 (ideally focusing the time of incorporation), and Weeks 5 and 9 (post-administration period). The intact compound and various (at least 4) hydroxy-metabolites exhibited similar elimination profiles. The peak urinary concentration (approximately 920 pg/ml) was observed after 8 h and is reduced to the detection limit (2 pg/ml) on Day 42 following administration of 760 µg GSK2881078. Correspondingly, hair concentrations of GSK2881078 (intact compound only) following a single oral dose of 1.5 mg GSK2881078 reached a peak concentration of 1.7 pg/mg in the segments collected 3 weeks post administration, representing the time of ingestion. The concentration rapidly declined to trace amounts of 0.7 (Week 5) and 0.2 pg/mg (Week 9), respectively. In conclusion, measurement of the intact compound GSK2881078 is feasible for both urine and hair analysis. However, concentrations in hair after single oral administration are in the low pg/mg range and can only be detected, if the segments cover the administration period.

In vitro metabolism of selected bioactive compounds of Eurycoma longifolia root extract to identify suitable markers in doping control.

Eurycoma longifolia Jack (Tongkat Ali, Simaroubaceae) is a medicinal plant endemic to South-East Asia. For centuries, different parts of the plant have been used as a natural remedy to treat fever, hypertension, or sexual insufficiency. Today, Eurycoma longifolia preparations are commercially available and advertised to enhance athletic performance and muscle strength. Several studies have demonstrated a testosterone-boosting effect that might be caused by the release of free testosterone from the sex-hormone-binding globulin. To date, many phytochemical constituents of Eurycoma longifolia root extracts have been identified and physiological effects have been examined, while studies on their biotransformation and monitoring are still lacking. Within this study, eurycomalide C, eurycomalactone, 5,6-dehydro-eurycomalactone, longilactone, 14,15ß-dihydroklaieanone, 11-dehydroklaieanone, 9-hydroxycanthin-6-one, and 9-methoxycanthin-6-one isolated from E. longifolia root were incubated with liver microsomes. Respective metabolites were analyzed by liquid chromatography-tandem (high-resolution) mass spectrometry. The compounds were chosen based on their potential androgenic effects (estimated by in vitro assays), their concentrations in plant extracts, and presumptive metabolic pathways. Hydroxylated phase I metabolites were only observed for 5,6-dehydro-eurycomalactone, 11-dehydroklaieanone, 9-hydroxycanthin-6-one, and 9-methoxycanthin-6-one. Moreover, an O-demethylated metabolite of 9-methoxycanthin-6-one was found. Besides, the glucuronide of 9-hydroxycanthin-6-one was detected after in vitro glucuronidation using liver microsomes. The in vitro generated metabolites were comparable to that detected in urine and serum after a single ingestion of either 9-methoxycanthin-6-one or an Eurycoma longifolia root extract. Hence, 9-methoxycanthin-6-one, its glucuronide, and the glucuronide of its O-demethylated biotransformation product are proposed to be the most suitable targets for detection of 9-methoxycanthin-6-one or Tongkat Ali application in urine and serum.

Statistical significance of hair analysis of clenbuterol to discriminate therapeutic use from contamination.

Clenbuterol is a well-established ß2-agonist, which is prohibited in sports and strictly regulated for use in the livestock industry. During the last few years clenbuterol-positive results in doping controls and in samples from residents or travellers from a high-risk country were suspected to be related the illegal use of clenbuterol for fattening. A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to detect low clenbuterol residues in hair with a detection limit of 0.02 pg/mg. A sub-therapeutic application study and a field study with volunteers, who have a high risk of contamination, were performed. For the application study, a total dosage of 30 µg clenbuterol was applied to 20 healthy volunteers on 5 subsequent days. One month after the beginning of the application, clenbuterol was detected in the proximal hair segment (0-1 cm) in concentrations between 0.43 and 4.76 pg/mg. For the second part, samples of 66 Mexican soccer players were analyzed. In 89% of these volunteers, clenbuterol was detectable in their hair at concentrations between 0.02 and 1.90 pg/mg. A comparison of both parts showed no statistical difference between sub-therapeutic application and contamination. In contrast, discrimination to a typical abuse of clenbuterol is apparently possible. Due to these findings results of real doping control samples can be evaluated.

Diagnostic value of concentration profiles of glucocorticosteroids and endocannabinoids in hair.

BACKGROUND: Endogenous corticosteroids and endocannabinoids are both known to be involved in stress adaption and anti-inflammatory and immuneregulatory effects. The application of hair as retrospective specimen for long-term recording of corticosteroids and its association with stress-induced biochemical alterations was intensively examined. METHODS: To evaluate the stability and correlation of various parameters of the endocannabinoid and corticosteroid systems, a prospective study was carried out. Hair samples were collected monthly over a pregnancy cycle (sixth week of pregnancy to 9 weeks postpartum). By comparison of hair concentrations in particular segments (ie, grown in the same time span but collected at different times), an examination of analyte stability in hair was achieved. Additionally, the comparison of proximal segments provided on biochemical information that is independent of alteration due to physical instability. The detection limits of a validated procedure using solid-phase extraction cleanup and liquid chromatography-mass spectrometry proved to be suitable to identify the endogenous levels of cortisol (limits of detection = 1.6 pg/mg), cortisone (2.1 pg/mg), anandamide (AEA, 0.3 pg/mg), and 2-arachidonoylglycerol (15 pg/mg). RESULTS: Corticosteroid concentrations in corresponding hair segments were found to be reduced with increasing hair age; an average decline of cortisol and cortisone by 50% in 4 months was estimated. Independently, an increase of cortisol and cortisone in proximal segments collected during pregnancy was confirmed, which is assumed to be stress related. Endocannabinoids proved to be by far more stable, as demonstrated by subsequent monthly collection of corresponding segments and there was hardly any washout of AEA detectable. Elevated hair concentrations of AEA and 2-arachidonoylglycerol were detected in the first-second trimester of pregnancy, which corresponds to negative correlations between AEA, cortisol, and cortisone.

Characterization of identity, metabolism and androgenic activity of 17-hydroxyandrosta-3,5-diene by GC-MS and a yeast transactivation system.

Anabolic-androgenic steroids are frequently misused compounds in sports, and they belong to the controlled substances according to the requirements of the World Anti-Doping Agency. The classical techniques of steroid detection are mass spectrometry coupled to gas or liquid chromatography. Biological methods that base on the ability of substances to bind the steroid receptor are not applied in routine doping control procedures so far, but they appear to be useful for characterization of steroid androgenic potential. In this study we used the yeast androgen receptor reporter system (YAS), which in the past has already successfully been applied to both various androgenic substances and also urine samples. Giving attention to the androgenic potential of steroidal dietary supplements, we exemplified the analysis using both mass spectrometry techniques and the YAS-based assay on the product "Syntrax Tetrabol" which was a confiscated dietary supplement and marketed as a steroid precursor. Identification, structure and the kinetic behavior of its excreted metabolites were analyzed by NMR, GC-MS and LC-MS/MS. The androgenic potential of the parent compound as well as its metabolites in urine was evaluated with the help of the YAS. The application of urine samples with a previous deconjugation and the inclusion of urine density values were carried out and led to increased responses on the YAS. Further, the possibility of a complementary application of structure-based instrumental analysis and biological detection of androgenicity with the help of the YAS seems to be desirable and is discussed.

Gene expression profiling in human whole blood samples after controlled testosterone application and exercise.

Doping with anabolic agents is regulated within a number of sports. Testosterone and its functional analogs are popular compounds for increasing muscle mass, physical performance, recovery, and reducing body fat. While routine tests for anabolic drugs exist (e.g. hair, urine, and blood analysis), the aim of the present study is to determine specific gene expression profiles (induced by testosterone and exercise) which may be used as effective biomarkers to determine the use of anabolic drugs. In this study, whole blood samples of 19 male volunteers were analyzed by semi-quantitative real-time polymerase chain reaction (RT-PCR) for gene expression profiles in the context of exercise and transdermal testosterone application (1.5 mg/kg body weight). The hormone application was monitored by urine and saliva analysis for testosterone. Both urinary and saliva levels indicate that transdermal testosterone application leads to an increase of testosterone, especially after exercise. RT-PCR results showed a clear variation in the expression of target genes as well as established housekeeping genes. Only one of the nine common housekeeping genes, cyclophilin b (PPIB), appears to be independent of both exercise and testosterone. Out of 14 candidate genes, five are unregulated; all others were more or less influenced by the mentioned variables. Only interleukin-6 appeared to be exclusively dependent on long-term testosterone application. This study indicates that many genes are not influenced by testosterone alone while exercise modulates gene expression in whole blood samples. As such, exercise must be considered when validating gene expression techniques for doping analysis.

Epidemiological investigation of the UGT2B17 polymorphism in doping control urine samples and its correlation to T/E ratios.

The deletion polymorphism of the enzyme UGT2B17 is known to correlate with the level of the testosterone to epitestosterone (T/E) ratio in urine specimen. Due to the importance of the T/E ratio to detect testosterone abuse in doping analysis, a PCR-ELISA system (Genotype® UGT test, AmplexDiagnostics) was established to identify the UGT2B17 phenotype in urine samples. Epidemiological investigations in a set of 674 routine doping controls (in- and out-of-competition) resulted in 22.8% homozygote gene-deleted and 74.5% UGT2B17-positive athletes. The validated test system has shown to be robust and sensitive: in only 18 cases (2.7%) isolation of cell material from urine failed. Following hydrolysis of glucuronidated conjugates, steroids were analyzed as bis-TMS derivatives by gas chromatography-mass spectrometry (GC-MS), for example, testosterone (T) and epitestosterone (E). Additionally, isotope ration mass spectrometry (IRMS) analysis and luteinizing hormone (LH) measurement were applied. Mean T/E ratios significantly correlated with the UGT2B17 phenotype (del: T/E 0.9; pos: 1.7), however the values did not differ as distinctive as reported in previous studies. Additionally, the T/E ratios in the gene-deleted group did not show a normal curve of distribution (median of T/E 0.5). Obviously, beside the UGT2B17 deletion further influences have to be taken into account, for example, polymorphisms or induction of other metabolizing enzymes. Our results indicate that the UGT2B17 polymorphism might be insufficient when utilized solely as a crucial parameter for individual interpretation of T/E in urine. Nevertheless, the detection of the UGT2B17-gene deletion in urine samples would provide additional information important for gathering evidence in analysis of steroids in doping control.

Ethylglucuronide as a potential marker for alcohol-induced elevation of urinary testosterone/epitestosterone ratios.

The potential influence of alcohol consumption on endogenous steroids has already been described in the literature. In those studies the ethanol level after ingestion was monitored using its concentration in blood, urine or saliva. Corresponding methods are not commonly used in anti-doping laboratories. Ethylglucuronide (EtG), which can be easily detected by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), appears to be a more suitable parameter for this purpose. It is slowly excreted into the urine and indicates alcohol intake for a much longer period than blood or urinary alcohol and it is therefore routinely used for legal purposes as an alcohol consumption marker. In pharmacokinetic studies that aimed to establish calculation models after ethanol intake, the formation of EtG was observed to coincide with elevated urinary testosterone/epitestosterone (T/E) ratios. Similarly, large amounts of EtG were correlated with abnormal steroid profiles found in routine doping samples. In this pilot study, several cases with significantly elevated T/E ratios were associated with urinary EtG concentrations higher than 50 microg/mL. These findings confirmed recent intake of ethanol in considerable amounts and suggest a connection to changes in specific steroid profile parameters. Owing to the ease with which procedures to determine EtG can be carried out, and the potential for such procedures to be introduced into screening schemes, the inclusion of this marker in the final evaluation of suspicious outliers in T/E ratio longitudinal studies would seem to be very useful.