Simultaneous quantitation and identification of intact Nandrolone phase II oxo-metabolites based on derivatization and inject LC-MS/(HRMS) methodology.

Α sensitive and selective derivatization and inject method for the quantification of intact nandrolone phase II oxo-metabolites was developed and validated using liquid chromatography - (tandem high resolution) mass spectrometry (LC-MS/(HRMS)). For the derivatization, Girard's reagent T (GRT) was used directly in natural urine samples and the analysis of the metabolites of interest was performed by direct injection into LC-MS/(HRMS) system operating in positive ionization mode. Derivatization enabled the efficient detection of nandrolone oxo-metabolites, while at the same time producing intense product ions under collision-induced dissociation (CID) conditions that are related to metabolites of the steroid backbone and not to the conjugated moieties. Glucuronide and sulfate metabolites of nandrolone were chromatographically resolved and quantified in the same run in the range of 1-100 ng mL-1, while at the same time structure identification could be performed for each metabolite. Full validation of the method was performed according to the World Anti-Doping Agency (WADA) International Standard for Laboratories (ISL). Nandrolone oxo-metabolites were quantified in two sets of urine samples, the first set consisted of real urine samples previously detected as negative and the second set consisted of urine samples collected from two excretion studies after nandrolone decanoate administration. The results for 19-norandrosterone glucuronide (19-NAG) and 19-noretiocholanolone glucuronide (19-NEG) were compared with those obtained by traditional gas chromatography - (tandem) mass spectrometry (GC-MS/[MS]) method.

New long-standing metabolites of 17α-methyltestosterone are detected in HepG2 cell in vitro metabolic model and in human urine.

Novel metabolites of the anabolic androgenic steroid 17α-methyltestosterone have been detected in HepG2 cell in vitro metabolic model and in human urine. Their detection was accomplished through targeted gas chromatography-(tandem) mass spectrometry analysis that has been based on microscale synthesized standards. The related synthesis and the gas chromatography-(tandem) mass spectrometry characterization of the analytical standards are described. All newly presented metabolites have a fully reduced steroid A-ring with either an 17,17-dimethyl-18-nor-Δ13 structure or they have been further oxidized at position 16 of the steroid backbone. Metabolites with 17,17-dimethyl-18-nor-Δ13 structure may be considered as side products of phase II metabolic sulfation of the 17ß-hydroxy group of methyltestosterone or its reduced tetrahydro-methyltestosterone metabolites 17α-methyl-5ß-androstane-3α,17ß-diol and 17α-methyl-5α-androstane-3α,17ß-diol that produce the known epimeric 17ß-methyl-5ß-androstane-3α,17α-diol and 17ß-methyl-5α-androstane-3α,17α-diol metabolites. The prospective of these new metabolites to increase detection time windows and improve identification was investigated by applying the World Anti-doping Agency TD2021IDCR criteria. The new metabolites, presented herein, complement the current knowledge on the 17α-methyltestosterone metabolism and in some cases can be used as additional long-term markers in the frame of sport doping drug testing.

LC-MS/(MS) confirmatory doping control analysis of intact phase II metabolites of methenolone and mesterolone after Girard's Reagent T derivatization.

In the present study, the application and evaluation of Girard's Reagent T (GRT) derivatization for the simultaneous detection and significantly important identification of different phase II methenolone and mesterolone metabolites by LC-MS/(MS) are presented. For the LC-MS analysis of target analytes two complementary isolation methods were developed; a derivatization and shoot method in which native urine is diluted with derivatization reagent and is injected directly to LC-MS and a liquid-liquid extraction method, using ethyl acetate at pH 4.5, for the effective isolation of both sulfate and glucuronide metabolites of the named steroids as well as of their free counterparts. For the evaluation of the proposed protocols, urine samples from methenolone and mesterolone excretion studies were analyzed against at least one sample from a different excretion study. Retention times, along with product ion ratios, were evaluated according to the WADA TD2021IDCR requirements, in order to determine maximum detection and identification time windows for each metabolite. Established identification windows obtained after LC-MS/(MS) analysis were further compared with those obtained after GC-MS/(MS) analysis of the same samples from the same excretion studies, for the most common analytes monitored by GC-MS/(MS). Full validation was performed for the developed derivatization and shoot method for the identification of methenolone metabolite, 3α-hydroxy-1-methylen-5α-androstan-17-one-3-glucuronide (mth3). Overall, the GRT derivatization presented herein offers a tool for the simultaneous sensitive detection of free, intact glucuronide and sulfate metabolites by LC-MS/(MS) that enhance significantly the detection and identification time windows of specific methenolone and mesterolone metabolites for doping control analysis.

Liquid chromatography-mass spectrometry behavior of Girard's reagent T derivatives of oxosteroid intact phase II metabolites for doping control purposes.

Intact phase II steroid metabolites have poor product ion mass spectra under collision-induced dissociation (CID) conditions. Therefore, we present herein the liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/(MS)) behavior of intact phase II metabolites of oxosteroids after derivatization. Based on the fact that Girard's reagent T (GRT), as derivatization reagent, was both convenient and efficient in terms of the enhancement in the ionization efficiency and the production of diagnostic product ions related to the steroid moiety, the latter was preferably selected between methoxamine and hydroxylamine upon the model compounds of androsterone glucuronide and androsterone sulfate. Sixteen different glucuronides and 29 sulfate conjugated metabolites of anabolic androgenic steroids (AASs), available either as pure reference materials or synthesized/extracted from administration studies, were derivatized with GRT, and their product ion spectra are presented. Product ion spectra include in all cases high number of product ions that in some cases are characteristic for certain structures of the steroid backbone. More specifically, preliminary results have shown major differences in fragmentation pattern for 17α/17ß-isomers of the sulfate conjugates, but limited differentiation for 17α/17ß-isomers of glucuronide conjugates and for 3α/3ß- and 5α/5ß-stereoisomers of both sulfate and glucuronide conjugates. Further to the suggestion of the current work, application on mesterolone administration studies confirmed-according to the World Anti-Doping Agency (WADA) TD2015IDCR-the presence of seven intact phase II metabolites, one glucuronide and six sulfates with use of LC-ESI-MS/(MS).

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.

Determination of anabolic androgenic steroids as imidazole carbamate derivatives in human urine using liquid chromatography-tandem mass spectrometry.

Anabolic androgenic steroids are widely abused substances in sports doping. Their detection present limitations regarding the use of soft ion sources such as electrospray or atmospheric pressure chemical ionization by liquid chromatography-tandem mass spectrometry. In the current study, a novel derivatization method was developed for the ionization enhancement of selected anabolic androgenic steroids. The proposed method aims at the introduction of an easily ionizable moiety into the steroid molecule by converting the hydroxyl groups into imidazole carbamates using 1,1'-carbonyldiimidazole as derivatization reagent. The proposed method was applied to water and urine samples spiked with exogenous anabolic androgenic steroids in various concentration levels. Steroid imidazole carbamate derivatives have shown intensive [M+H]+ signals under electrospray ionization and common fragmentation patterns in tandem mass spectrometry mode with [M-CO2 +H]+ and [M-ΙmCO2 +H]+ as major ions with low collision energy. The obtained results showed that the majority of steroids were detectable at concentrations equal or lower to their minimum required performance level according to the World Anti-Doping Agency technical document. The proposed method is sensitive with a preparation procedure that could be easily applied to the analysis of doping control samples.

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.

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.

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.

Examination of the kinetic isotopic effect to the acetylation derivatization for the gas chromatographic-combustion-isotope ratio mass spectrometric doping control analysis of endogenous steroids.

In gas chromatographic-combustion-isotope ratio mass spectrometry (GC-C-IRMS) doping control analysis, endogenous androgenic anabolic steroids and their metabolites are commonly acetylated using acetic anhydride reagent, thus incorporating exogenous carbon that contributes to the measured isotope ratio. Comparison of the endogenous δ(13)C of free, mono-, and di-acetylated steroids requires application of corrections, typically through straightforward use of the mass balance equation. Variability in kinetic isotope effects (KIE) due to steroid structures could cause fractionation of endogenous steroid carbon, resulting in inaccurate results. To test for possible KIE influence on δ(13)C, acetic anhydride of graded isotope ratio within the natural abundance range was used under normal derivatization conditions to test for linearity. In all cases, plots of measured steroid acetate δ(13)C versus acetic anhydride δ(13)C were linear and slopes were not significantly different. Regression analysis of the Δδ(13)C of enriched acetic anhydrides versus Δδ(13)C of derivatized steroids shows that KIE are similar in all cases. We conclude that δ(13)C calculated from the mass balance equation is independent of the δ(13)C of the acetic anhydride reagent, and that net KIE under normal derivatization conditions do not bias the final reported steroid δ(13)C.

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.