Searching for in silico predicted metabolites and designer modifications of (cortico)steroids in urine by high-resolution liquid chromatography/time-of-flight mass spectrometry.

Glucocorticosteroids are a restricted class of substances and appear on the 'in-competition' prohibited list of the World Anti-Doping Agency (WADA). Analysis of glucocorticosteroids is complicated since they show significant phase 1 and 2 metabolism in the human body and are excreted into urine in concentrations in the microg/L range. Full scan, high-resolution time-of-flight mass spectrometry analysis generates information on all ionisable components in urine, including known and unknown metabolites of steroids and even designer modifications of anabolic steroids. However, evaluation of the data obtained can be difficult and time-consuming because of the need to differentiate between endogenous components and compounds of interest. MetaboLynx, a spectral and chromatographic search program, was modified for the determination of in silico predicted metabolites of glucocorticosteroids and designer modifications of anabolic steroids in human urine. Spiked urine samples were successfully screened for known components in a targeted approach and for unknown species in a non-targeted approach using data filtering to limit potential false-positives. A simplified combined approach of targeted and untargeted screening was used for the detection of metabolites and designer modifications of existing compounds. This approach proved successful and showed its strength in the detection of tetrahydrogestrinone (THG), a designer modification of gestrinone. THG was positively detected in a spiked urine sample and correctly identified as a twofold hydrogenation of gestrinone. The developed screening method can easily be adapted to specific needs and it is envisaged that a similar approach would be amendable to the discovery of metabolites or designer modifications of other compounds of interest.

Detection and pharmacokinetics of tetrahydrogestrinone in horses.

The anti-doping rules of national and international sport federations ban any use of tetrahydrogestrinone (THG) in human as well as in horse sports. Initiated by the THG doping scandals in human sports a method for the detection of 3-keto-4,9,11-triene steroids in horse blood and urine was developed. The method comprises the isolation of the analytes by a combination of solid phase and liquid-liquid extraction after hydrolysis and solvolysis of the steroid conjugates. The concentrations of THG in blood and urine samples were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A THG excretion study on horses was conducted to verify the method capability for the analysis of postadministration urine samples. In addition, blood samples were collected to allow for determination of the pharmacokinetics of THG in horses. Following the administration of a single oral dose of 25 microg THG per kg bodyweight to 10 horses, samples were collected at appropriate intervals. The plasma levels of THG reached maximal concentrations of 1.5-4.8 ng/mL. Twenty-four hours after the administration plasma levels returned to baseline. In urine, THG was detectable for 36 h. Urinary peak concentrations of total THG ranged from 16 to 206 ng/mL. For the 10 horses tested, the mean plasma clearance of THG was 2250 mL/h/kg and the plasma elimination half-life was 1.9 h.

Screening of in vitro synthesised metabolites of 4,9,11-trien-3-one steroids by liquid chromatography mass spectrometry.

The aim of the work was to develop a flexible in vitro synthesis procedure, which can be applied in order to study and predict the metabolic patterns of new derivatives of anabolic androgenic steroids (AAS) with respect to most prominent target compounds for doping control purposes. Microsomal and S9 fraction of human liver preparations were used as a source of metabolising enzymes and the co-substrates of the synthesis mixture were selected to favour phase-I metabolic reactions and glucuronidation as phase-II conjugation reactions. Model compounds within the study were 4,9,11-trien-3-one steroids, structural derivatives of gestrinone and trenbolone, which both are included in the list of prohibited compounds in sports by the World Anti-Doping Agency (WADA). The correlation between in vitro metabolism of human microsomes and in vivo excretion studies in human was compared with gestrinone and subsequently, the applicability of the in vitro model for prediction of AAS metabolic pathways for new doping agents was evaluated. All the AAS examined within this study were successfully metabolised using the developed in vitro model, hydroxylation, reduction and glucuronide conjugation being the most prominent reaction pathways. Hydroxylated and glucuronide-conjugated metabolites of in vivo experiment with gestrinone were the same metabolites formed in the enzyme-driven process, thus showing good in vitro-in vivo correlation. Liquid chromatographic-mass spectrometric and tandem mass spectrometric methods were developed, relying on the positive polarity of electrospray ionisation, which also allowed the direct detection of intact glucuronide-conjugated AAS metabolites. Due to charge delocalisation and high proton affinity, the developed method was proven effective in the analysis of AAS metabolites bearing extensive conjugated double bond systems in their structures.

Detection of anabolic steroid abuse using a yeast transactivation system.

The classical analytical method for detection of anabolic steroid abuse is gas chromatography followed by mass spectrometry (GC/MS). However, even molecules with a chemical structure typical for this class of substances, are sometimes not identified in routine screening by GC/MS when their precise chemical structure is still unknown. A supplementary approach to identify anabolic steroid abuse could be a structure-independent identification of anabolic steroids based on their biological activity. To test the suitability of such a system, we have analyzed the yeast androgen receptor (AR) reporter gene system to identify anabolic steroids in human urine samples. Analysis of different anabolic steroids dissolved in buffer demonstrated that the yeast reporter gene system is able to detect a variety of different anabolic steroids and their metabolites with high specificity, including the so-called 'designer steroid' tetrahydrogestrinone. In contrast, other non-androgenic steroids, like glucocordicoids, progestins, mineralocordicoids and estrogens had a low potency to stimulate transactivation. To test whether the system would also allow the detection of androgens in urine, experiments with spiked urine samples were performed. The androgen reporter gene in yeast responds very sensitive to 5alpha-dihydrotestosterone (DHT), even at high urine concentrations. To examine whether the test system would also be able to detect anabolic steroids in the urine of anabolic steroid abusers, anonymous urine samples previously characterized by GCMS were analyzed with the reporter gene assay. Even when the concentration of the anabolic metabolites was comparatively low in some positive samples it was possible to identify the majority of positive samples by their biological activity. In conclusion, our results demonstrate that the yeast reporter gene system detects anabolic steroids and corresponding metabolites with high sensitivity even in urine of anabolic steroid abusing athletes. Therefore we believe that this system can be developed towards a powerful (pre) screening tool for the established doping tests. The system is easy to handle, robust, cost-efficient and needs no high-tech equipment. But most importantly, a biological test system does not require knowledge of the chemical structure of androgenic substances and therefore suitable to detect previously unidentified substances, especially those of the class of so-called designer steroids.

Preparation of antibodies for the designer steroid tetrahydrogestrinone and development of an enzyme-linked immunosorbent assay for human urine analysis.

A highly sensitive enzyme-linked immunosorbent assay (ELISA) has been developed for tetrahydrogestrinone (THG), the new designer anabolic steroid responsible for the well-known Balco scandal announced in year 2003. Antibodies have been raised against 18a-homo-pregna-4,9,11-trien-17beta-ol-3-carboxymethyl oxime coupled to horseshoe crab hemocyanin. The hapten has been synthesized from gestrinone by controlled reduction of the triple bond of the ethinyl group at position C-17, without affecting the double bonds of the steroidal rings, followed by reaction of the keto group at C-3 with (carboxymethoxy)amine hemihydrochloride to form the oxime bond. The antisera obtained has been used in combination with 18a-homo-pregna-4,9,11-trien-17beta-ol-20-yn-3-carboxymethyl oxime, a hapten derivative of gestrinone, coupled to bovine serum albumin to establish a competitive ELISA. Under the conditions used, THG can be detected in buffer with a limit of detection (LOD) of 0.045 +/- 0.015 microg L(-1) (N = 9). The assay is very selective since other steroids assessed are not recognized. Preliminary experiments performed with human urine samples demonstrate that the assay can be applied to the analysis of these samples after a simple sample treatment method reaching a LOD of 0.25 +/- 0.14 microg L(-1). Accuracy is very good as demonstrated by the excellent correlation obtained when analyzing blind spiked urine samples (slope 0.93, R2 = 0.992).

Analysis of synthetic 19-norsteroids trenbolone, tetrahydrogestrinone and gestrinone by gas chromatography-mass spectrometry.

Tetrahydrogestrinone, gestrinone and trenbolone are synthetic 19-norsteroids with androgenic properties sharing a labile conjugated ketotrienyl motif. Their LC-MS analyses tend to overcome classical derivatization problems, a shortcoming to the use of GC-MS. Therefore, alternative derivatization procedures were evaluated. The procedure with methoxylamine: pyridine followed by TMSImid: MSTFA gave the best results. This is attributed to the stability of the MO-TMS derivatives hindering the formation of artifacts and tautomerism. A full method is presented including SPE, hydrolysis and liquid-liquid extraction. It was possible to confirm the analytes below 2 ng/mL in urine, being the method robust and cost effective also for screening proposes.

Urine testing for designer steroids by liquid chromatography with androgen bioassay detection and electrospray quadrupole time-of-flight mass spectrometry identification.

New anabolic steroids show up occasionally in sports doping and in veterinary control. The discovery of these designer steroids is facilitated by findings of illicit preparations, thus allowing bioactivity testing, structure elucidation using NMR and mass spectrometry, and final incorporation in urine testing. However, as long as these preparations remain undiscovered, new designer steroids are not screened for in routine sports doping or veterinary control urine tests since the established GC/MS and LC/MS/MS methods are set up for the monitoring of a few selected ions or MS/MS transitions of known substances only. In this study, the feasibility of androgen bioactivity testing and mass spectrometric identification is being investigated for trace analysis of designer steroids in urine. Following enzymatic deconjugation and a generic solid-phase extraction, the samples are analyzed by gradient LC with effluent splitting toward two identical 96-well fraction collectors. One well plate is used for androgen bioactivity detection using a novel robust yeast reporter gene bioassay yielding a biogram featuring a 20-s time resolution. The bioactive wells direct the identification efforts to the corresponding well numbers in the duplicate plate. These are subjected to high-resolution LC using a short column packed with 1.7-microm C18 material and coupled with electrospray quadrupole time-of-flight mass spectrometry (LC/QTOFMS) with accurate mass measurement. Element compositions are calculated and used to interrogate electronic substance databases. The feasibility of this approach for doping control is demonstrated via the screening of human urine samples spiked with the designer anabolic steroid tetrahydrogestrinone. Application of the proposed methodology, complementary to the established targeted urine screening for known anabolics, will increase the chance of finding unknown emerging designer steroids, rather then being solely dependent on findings of the illicit preparations themselves.

Discovery, biosynthesis, and structure elucidation of metabolites of a doping agent and a direct analogue, tetrahydrogestrinone and gestrinone, using human hepatocytes.

Tetrahydrogestrinone (18a-homo-pregna-4,9,11-trien-17beta-ol-3-one, THG) is an anabolic androgenic steroid sold to athletes as an undetectable performance enhancer. Being an unapproved substance, no legitimate in vivo human excretion studies could be performed to identify urinary markers of this doping agent. In vitro systems were used as an alternative approach to study the human metabolism of THG and the gestrinone analogue, which is a marketed drug. Incubations of both compounds in the presence of human hepatocytes led to formation of oxidative and glucuroconjugated metabolites. Microgram quantities of the major in vitro metabolites were biosynthesized using human hepatocytes, characterized by HPLC/MS/MS, and their structures elucidated by NMR. Due to high structure similarity, both THG and gestrinone had an analogous in vitro metabolic pathway leading to successive addition of a hydroxyl and a beta-glucuronic acid at C-18. This in vitro metabolite of gestrinone was consistent with a previously reported major but unknown human urinary metabolite. The structure of another metabolite of THG was proposed to be a glucuroconjugate of an oxidative product with a hydroxyl group most likely at C-16epsilon. In vitro information reported therein could significantly impact the identification of new urinary markers of THG for doping control purposes.

Tetrahydrogestrinone: discovery, synthesis, and detection in urine.

Tetrahydrogestrinone (18a-homo-pregna-4,9,11-trien-17beta-ol-3-one or THG) was identified in the residue of a spent syringe that had allegedly contained an anabolic steroid undetectable by sport doping control urine tests. THG was synthesized by hydrogenation of gestrinone and characterized by mass spectrometry and NMR spectroscopy. We developed and evaluated sensitive and specific methods for rapid screening of urine samples by liquid chromatography/tandem mass spectrometry (LC/MS/MS) of underivatized THG (using transitions m/z 313 to 241 and 313 to 159) and gas chromatography/high-resolution mass spectrometry (GC/HRMS) analysis of the combination trimethylsilyl ether-oxime derivative of THG (using fragments m/z 240.14, 254.15, 267.16, and 294.19). A baboon administration study showed that THG is excreted in urine.

Determination of the metabolites of gestrinone in human urine by high performance liquid chromatography, liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry.

Gestrinone was studied by high performance liquid chromatography (HPLC) for screening and by gas chromatography/mass spectrometry (GC/MS) for confirmation. When the chromatograms of blank, spiked urine and dosed urine were compared by HPLC, two unknown metabolites were found and these were excreted as the conjugated forms. Metabolites 1 and 2 were tested by LC/MS and LC/MS/MS and both had parent ions at m/z 325. The fragment ion of metabolite 1 was at m/z 263 and ions for metabolite 2 were m/z 307 [MH - H(2)O](+), 289, 279 and 241. LC/MS/MS of m/z 263 as the parent ion of metabolite 1 gave fragment ions at m/z 245 and 217, which were assumed to be [263 - H(2)O](+) and [235 - H(2)O](+), respectively. The trimethylsilyl (TMS)-enol-TMS ether derivative of gestrinone displayed three peaks in its GC/MS chromatogram, formed by tautomerism.

Determination and excretion study of gestrinone in human urine by high performance liquid chromatography and gas chromatography/mass spectrometry.

Gestrinone was studied by HPLC for screening and by GC/MS for confirmation. Three unknown peaks were found by HPLC which are probably the metabolites of gestrinone, and conjugated gestrinone in dosed human urine. The metabolites and gestrinone were excreted as the conjugated forms. The total amounts of metabolite 1 and conjugated gestrinone, recovered after 48 h, were 0.20 and 0.32 mg, respectively. When metabolite 1 was tested by LC/MS and LC/MS/MS, the parent ion was m/z 327, [MH](+), and fragment ions were seen at m/z 309 [MH - H(2)O](+), 291 [MH - 2H(2)O](+), 283, 263 and 239. The TMS-enol-TMS ether derivative of gestrinone has three peaks in the GC/MS chromatogram formed by tautomerism. The reproducibility of the derivatization method was stable and recoveries were over 87% when spiked into blank urine.