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.

Alternative markers for Methylnortestosterone misuse in human urine.

Methylnortestosterone is a progestin and synthetic androgenic anabolic steroid, prohibited by WADA. Methylnortestosterone misuse is commonly detected by monitoring the parent compound and its main metabolites, 17α-methyl-5α-estrane-3α, 17ß-diol (M1) and 17α-methyl-5ß-estrane-3α, 17ß-diol (M2), in the glucuronide fraction. In the current study, a direct detection of methylnortestosterone sulfo-conjugated metabolites after ethyl acetate extraction and analysis by LC/Q/TOF-MS in negative ionization mode was performed, detecting two main sulfate metabolites (S1, S2). For the characterization of metabolites, samples from the excretion study, were additionally analyzed by GC-MS, after solvolysis and per TMS derivatization. RT and MS data collected, were compared with RT and MS data from metabolites of 17z-methyl-5α/ß-estrane-3α/ß, 17z-diols structures with prefixed stereochemistry at 3 and 5 positions, synthesized through Grignard reaction from 19-noretiocholanolone, 19-norandrosterone and 19-norepiandrosterone. Confirmed sulfate metabolites were S1, 17α-methyl-5α-estrane-3α, 17ß-diol 3α sulfate (detected up to 72 h) and S2, 17α-methyl-5ß-estrane-3α, 17ß-diol 3α sulfate (detected up to 192 h). Furthermore, applying targeted analysis based on RT and MS data of the synthesized metabolites two additional metabolites M3, 17ß-methyl-5ß-estrane-3α, 17α-diol and M4, 17ß-methyl-5α-estrane-3α, 17α-diol were detected in the glucuronide fraction and one more metabolite (S3) 17ß-methyl-5ß-estrane-3α, 17α-diol was detected in the sulfate fraction in lower abundance until the end of the excretion study (192 h). Interestingly, S2 could also be detected after the direct analysis of non-hydrolyzed steroid by GC-MS/MS as artifact, following normal ProcIV anabolic steroid procedure and using diethylether as extraction solvent.

Gas chromatographic quadrupole time-of-flight full scan high resolution mass spectrometric screening of human urine in antidoping analysis.

This paper presents the development and validation of a high-resolution full scan (FS) electron impact ionization (EI) gas chromatography coupled to quadrupole Time-of-Flight mass spectrometry (GC/QTOF) platform for screening anabolic androgenic steroids (AAS) in human urine samples. The World Antidoping Agency (WADA) enlists AAS as prohibited doping agents in sports, and our method has been developed to comply with the qualitative specifications of WADA to be applied for the detection of sports antidoping prohibited substances, mainly for AAS. The method also comprises of the quantitative analysis of the WADA's Athlete Biological Passport (ABP) endogenous steroidal parameters. The applied preparation of urine samples includes enzymatic hydrolysis for the cleavage of the Phase II glucuronide conjugates, generic liquid-liquid extraction and trimethylsilyl (TMS) derivatization steps. Tandem mass spectrometry (MS/MS) acquisition was applied on few selected ions to enhance the specificity and sensitivity of GC/TOF signal of few compounds. The full scan high resolution acquisition of analytical signal, for known and unknown TMS derivatives of AAS provides the antidoping system with a new analytical tool for the detection designer drugs and novel metabolites, which prolongs the AAS detection, after electronic data files' reprocessing. The current method is complementary to the respective liquid chromatography coupled to mass spectrometry (LC/MS) methodology widely used to detect prohibited molecules in sport, which cannot be efficiently ionized with atmospheric pressure ionization interface.

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.

Analytical progresses of the World Anti-Doping Agency Olympic laboratories: a 2016 update from London to Rio.

The 2016 Olympic and Paralympic Games, the biggest event in human sports, was held in Rio de Janeiro with more than 10,500 athletes from 206 countries over the world competing for the highest of sports honors, an Olympic medal. With the hope that the Olympic ideal accompanies all aspects of the XXXI Olympiad, WADA accredited antidoping laboratories use the spearhead of analytical technology as a powerful tool in the fight against doping. This review summarizes the main analytical developments applied in antidoping testing methodology combined with the main amendments on the WADA regulations regarding analytical testing starting from the 2012 London Olympics until the 2016 Olympic Games in Rio de Janeiro.

Metabolism of isometheptene in human urine and analysis by gas chromatography-mass spectrometry in doping control.

A study of the metabolism of isometheptene, an antispasmodic drug, in man and comparison with heptaminol metabolism, is presented in this paper. Isometheptene and two metabolites were detected in human urine after oral administration of a tablet containing isometheptene mucate. The urine level of the parent drug, which is excreted during the first 24 h, was determined using gas chromatography-mass spectrometry, after alkaline extraction with organic solvent. A minor metabolite of isometheptene was converted to heptaminol in vitro under the acidic hydrolysis conditions used for the screening procedure of stimulants and narcotics in doping control analysis.

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

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

Gas chromatographic-mass spectrometric quantitation of busulfan in human plasma for therapeutic drug monitoring: a new on-line derivatization procedure for the conversion of busulfan to 1,4-diiodobutane.

A simplified gas chromatographic-mass spectrometric (GC-MS) analytical method, involving a novel derivatization procedure was developed for monitoring busulfan (Bu) plasma concentrations in populations undergoing bone marrow transplantation. Plasma samples (500 µL) containing Bu-d8 as internal standard were extracted with ethyl acetate (2 mL) followed by centrifugation (1800 rpm, 5 min) and evaporation of the organic layer under nitrogen flow (50 °C). The dry residue was reconstituted with 100 µL iodine solution in acetonitrile (0.25%, w/v) and 3 µL were injected into the GC-MS system at 250 °C. Conversion of Bu to 1,4-diiodobutane was accomplished on-line without the need of an extra derivatization step. MS was operated at selected ion monitoring mode at m/z 183 and 191 corresponding to Bu and Bu-d8 derivatives. Total analysis time was 11.5 min. Calibration curves were linear (mean r=0.9996) over a concentration range of 25-3651 ng/mL using a (1/x)-weighted scheme. Limit of detection and lower limit of quantitation were 10.6 and 25 ng/mL, respectively. Overall accuracy Er (%) was ranging from -5.10% to 10.5%. Within- and between-run RSD (%) were lower 4.51% and 2.15%, respectively. Overall recovery of Bu was equal to 69.3±4.56% (RSD (%)). The present method is sensitive and specific, requiring a simple sample preparation procedure and short analysis time, advantages crucial for therapeutic drug monitoring of Bu in clinical practice and application in pharmacokinetic studies.

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.

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 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.

Preventive doping control analysis: liquid and gas chromatography time-of-flight mass spectrometry for detection of designer steroids.

A new combined doping control screening method for the analysis of anabolic steroids in human urine using liquid chromatography/electrospray ionization orthogonal acceleration time-of-flight mass spectrometry (LCoaTOFMS) and gas chromatography/electron ionization orthogonal acceleration time-of-flight mass spectrometry (GCoaTOFMS) has been developed in order to acquire accurate full scan MS data to be used to detect designer steroids. The developed method allowed the detection of representative prohibited substances, in addition to steroids, at concentrations of 10 ng/mL for anabolic agents and metabolites, 30 ng/mL for corticosteroids, 500 ng/mL for stimulants and beta-blockers, 250 ng/mL for diuretics, and 200 ng/mL for narcotics. Sample preparation was based on liquid-liquid extraction of hydrolyzed human urine, and the final extract was analyzed as trimethylsilylated derivatives in GCoaTOFMS and underivatized in LCoaTOFMS in positive ion mode. The sensitivity, mass accuracy, advantages and limitations of the developed method are presented.