Determination of salmeterol, α-hydroxysalmeterol and fluticasone propionate in human urine and plasma for doping control using UHPLC-QTOF-MS.

Salmeterol and fluticasone are included in the Prohibited List annually issued by the World Anti-Doping Agency. While for other permitted beta-2 agonists a threshold has been established, above which any finding constitutes an Adverse Analytical Finding, this is not the case with salmeterol. The salmeterol metabolite, α-hydroxysalmeterol, has been described as a potentially more suitable biomarker for the misuse of inhaled salmeterol. In this study, a new and rapid UHPLC-QTOF-MS method was developed and validated for the simultaneous quantification of salmeterol, α-hydroxysalmeterol and fluticasone in human urine and plasma, which can be used for doping control. The analytes of interest were extracted by means of solid phase extraction and were separated on a Zorbax Eclipse Plus C18 column. Detection was performed in a quadrupole time-of-flight mass spectrometer equipped with an electrospray ionization source, in positive mode for the detection of salmeterol and its metabolite and in negative mode for the detection of fluticasone. Method was validated over a linear range from 0.10 to 2.00 ng/ml for salmeterol and fluticasone, and from 1.00 to 20.0 ng/ml for α-hydroxysalmeterol, in urine, whereas in plasma, the linear range was from 0.025 to 0.500 ng/ml for salmeterol and fluticasone, respectively.

Human in vivo metabolism study of LGD-4033.

Selective androgen receptor modulators (SARMs) are an emerging class of therapeutics targeted to cachexia, sarcopenia, and hypogonadism treatment. LGD-4033 is a SARM which has been included on the Prohibited List annually released by the World Anti-Doping Agency (WADA). The aim of the present work was the investigation of the metabolism of LGD-4033 in a human excretion study after administration of an LGD-4033 supplement, the determination of the metabolites' excretion profiles with special interest in the determination of its long-term metabolites, and the comparison of the excretion time of the phase I and phase II metabolites. The results were also compared to those derived from previous LGD-4033 studies concerning both in vitro and in vivo experiments. Supplement containing LGD-4033 was administered to one human male volunteer and urine samples were collected up to almost 21 days. Analysis of the hydrolyzed (with ß-glucuronidase) as well as of the non-hydrolyzed samples was performed using liquid chromatography-high resolution mass spectrometry (LC-HRMS) in negative ionization mode and revealed that, in both cases, the two isomers of the dihydroxylated metabolite (M5) were preferred target metabolites. The gluco-conjugated parent LGD-4033 and its gluco-conjugated metabolites M1 and M2 can be also considered as useful target analytes in non-hydrolyzed samples. The study also presents two trihydroxylated metabolites (M6) identified for the first time in human urine; one of them was recently reported in an LGD-4033 metabolism study in horse urine and plasma.

Doping control container for urine stabilization: a pilot study.

Urine collection containers used in the doping control collection procedure do not provide a protective environment for urine, against degradation by microorganisms and proteolytic enzymes. An in-house chemical stabilization mixture was developed to tackle urine degradation problems encountered in human sport samples, in cases of microbial contamination or proteolytic activity. The mixture consists of antimicrobial substances and protease inhibitors for the simultaneous inactivation of a wide range of proteolytic enzymes. It has already been tested in lab-scale, as part of World Anti-Doping Agency's (WADA) funded research project, in terms of efficiency against microbial and proteolytic activity. The present work, funded also by WADA, is a follow-up study on the improvement of chemical stabilization mixture composition, application mode and limitation of interferences, using pilot urine collection containers, spray-coated in their internal surface with the chemical stabilization mixture. Urine in plastic stabilized collection containers have been gone through various incubation cycles to test for stabilization efficiency and analytical matrix interferences by three WADA accredited Laboratories (Athens, Ghent, and Rome). The spray-coated chemical stabilization mixture was tested against microorganism elimination and steroid glucuronide degradation, as well as enzymatic breakdown of proteins, such as intact hCG, recombinant erythropoietin and small peptides (GHRPs, ipamorelin), induced by proteolytic enzymes. Potential analytical interferences, observed in the presence of spray-coated chemical stabilization mixture, were recorded using routine screening procedures. The results of the current study support the application of the spray-coated plastic urine container, in the doping control collection procedure. Copyright © 2016 John Wiley & Sons, Ltd.

Advances in the detection of designer steroids in anti-doping.

The abuse of unknown designer androgenic anabolic steroids (AAS) is considered to be an issue of significant importance, as AAS are the choice of doping preference according to World Anti-doping Agency statistics. In addition, unknown designer AAS are preferred since the World Anti-doping Agency mass spectrometric identification criteria cannot be applied to unknown molecules. Consequently, cheating athletes have a strong motive to use designer AAS in order to both achieve performance enhancement and to escape from testing positive in anti-doping tests. To face the problem, a synergy is required between the anti-doping analytical science and sports anti-doping regulations. This Review examines various aspects of the designer AAS. First, the structural modifications of the already known AAS to create new designer molecules are explained. A list of the designer synthetic and endogenous AAS is then presented. Second, we discuss progress in the detection of designer AAS using: mass spectrometry and bioassays; analytical data processing of the unknown designer AAS; metabolite synthesis; and, long-term storage of urine and blood samples. Finally, the introduction of regulations from sports authorities as preventive measures for long-term storage and reprocessing of samples, initially reported as negatives, is discussed.

A synopsis of the adverse analytical and atypical findings between 2005 and 2011 from the Doping Control Laboratory of Athens in Greece.

This article concerns the analysis of the Adverse Analytical Findings (AAFs) and the appropriate alterations made during the period 2005-2011, so that the Doping Control Laboratory of Athens (DCLA) obeys the updated World Anti-Doping Agency (WADA) List of Prohibited Substances. The % AAFs of the DCLA was compared with those of WADA-Accredited Laboratories. In 2008, the term Atypical Finding was introduced by the WADA representing a reported but inconclusive result. A characteristic example is when a testosterone-to-epitestosterone ratio is >4 followed by a negative gas chromatography/combustion/isotope ratio mass spectrometry result. In a total of about 30,000 athlete samples, 136 athletes were found with an increased testosterone/epitestosterone ratio and 43 with tetrahydrocannabinol metabolite (THCCOOH) of 427 reported AAFs. Twenty-one athletes in total were found positive with methylhexaneamine, the 11 found after a batch of 1000 samples was reprocessed. Besides, there were AAFs below their Minimum Required Performance Level (MRPL). The increasing need for higher detectability imposed new apparatus, e.g., liquid chromatography/quadrupole/time-of-flight mass spectrometry, whereas that for lowering the capital costs and reporting times led to the unification of the screening method which includes stimulants, diuretics, anabolics and other substances.

A generic screening methodology for horse doping control by LC-TOF-MS, GC-HRMS and GC-MS.

In the present study a general screening protocol was developed to detect prohibited substances and metabolites for doping control purposes in equine sports. It was based on the establishment of a unified sample preparation and on the combined implementation of liquid and gas chromatographic MS analysis. The sample pretreatment began with two parallel procedures: enzymatic hydrolysis of sulfate and glucuronide conjugates, and methanolysis of the 17ß-sulfate steroid conjugates. The extracts were treated for LC-TOF-MS, GC-HRMS and GC-MS assays. The majority of the prohibited substances were identified through a high mass accuracy technique, such as LC-TOF-MS, without prior derivatization. The sample preparation procedure included the formation of methylated and trimethylsilylated derivatives common in toxicological GC-MS libraries. The screening method was enhanced by post-run library searching using automated mass spectral deconvolution and identification system (AMDIS) combined with deconvolution reporting software (DRS). The current methodology is able to detect the presence of more than 350 target analytes in horse urine and may easily incorporate a lot of new substances without changes in chromatography. The full scan acquisition allows retrospective identification of prohibited substances in stored urine samples after reprocessing of the acquired data. Validation was performed for sixty representative compounds and included limit of detection, matrix interference - specificity, extraction recovery, precision, mass accuracy, matrix effect and carry over contamination. The suitability of the method was demonstrated with previously declared positive horse urine samples.

Stabilization of human urine doping control samples: a current opinion.

Transportation of doping control urine samples from the collection sites to the World Anti-doping Agency (WADA) Accredited Laboratories is conducted under ambient temperatures. When sample delivery is not immediate, microbial contamination of urine, especially in summer, is a common phenomenon that may affect sample integrity and may result in misinterpretation of analytical data. Furthermore, the possibility of intentional contamination of sports samples during collection with proteolytic enzymes, masking the abuse of prohibited proteins such as erythropoietin (EPO) and peptide hormones, is a practice that has already been reported. Consequently, stabilization of urine samples with a suitable method in a way that protects samples' integrity is important. Currently, no stabilization method is applied in the sample collection equipment system in order to prevent degradation of urine compounds. The present work is an overview of a study, funded by WADA, on degradation and stabilization aspects of sports urine samples against the above threats of degradation. Extensive method development resulted in the creation of a mixture of chemical agents for the stabilization of urine. Evaluation of results demonstrated that the stabilization mixture could stabilize endogenous steroids, recombinant EPO, and human chorionic gonadotropin in almost the entire range of the experimental conditions tested.

Stabilization of human urine doping control samples: IV. Human chorionic gonadotropin.

The presence of proteolytic enzymes in urine samples, coming from exogenous or endogenous sources, enhances the cleavage of human chorionic gonadotropin (hCG). Moreover, elevated temperatures occurring occasionally during the delayed transportation of sport urine samples, favor the nicking of the hCG molecule. The aim of the current study, funded by the World Anti-Doping Agency (WADA), was the application of a stabilization mixture in athletes' urine samples to chemically inactivate proteolytic enzymes coming from exogenous or endogenous sources so as to prevent the degradation of hCG. The stabilization mixture applied, already tested for the stabilization of endogenous steroids and recombinant erythropoietin (rEPO), was a combination of antibiotics, antimycotic substances, and protease inhibitors. Incubation experiments were conducted in the presence or absence of the stabilization mixture in urine aliquots spiked with six proteases (first series of experiments) and one microorganism associated with urinary tract infections (UTI) (second series of experiments). Intact hCG levels were evaluated by using the EIAgen Total hCG kit. In the first series of experiments, hCG levels were reduced in the untreated aliquots following incubation at 37 degrees C. The addition of the chemical stabilization mixture prevented degradation of hCG induced by four of the proteases applied. In the second series of experiments, no significant difference was found in urine inoculated with E. coli, between aliquots treated with chemical mixture and the untreated aliquots. The addition of the proposed chemical stabilization mixture improves the quality of athletes' urine samples against possible deterioration due to high temperatures or attempts of proteolytic manipulation.