Detection time comparison of non-hydrolysed sulphated metabolites of metenolone, mesterolone and 17α-methyltestosterone analysed by four different mass spectrometric techniques.

The frequent detection of anabolic androgenic steroids (AAS) indicates their popularity among rule-breaking athletes. The so called long-term metabolites play a crucial role in their detection, and non-hydrolysed sulphated metabolites have gained renewed interest, as research has demonstrated their extended detection time compared to the more conventional markers (e.g., for metenolone and mesterolone). Their potential has been investigated using liquid and gas chromatography-mass spectrometry (LC- and GC-MS). However, due to their complementary nature, chances are that the most promising metabolite on one technique does not necessarily exhibit the same behaviour on the other and vice versa. Therefore, a comparison was carried out where as a trial model, metenolone, mesterolone and 17α-methyltestosterone were selected and the most likely long-term sulphated metabolites identified on four mass spectrometric instruments. Additionally, using a modified sample preparation procedure, comparison between conventional and non-hydrolysed sulphated metabolites between different GC-MS instruments was also included. When focusing on each individual marker, no cases were observed where a single metabolite provided a superior detection time on all instruments. Furthermore, for each AAS, there were incidences where a metabolite provided the best detection time on one instrument but could only be detected for a shorter period or not at all on other instruments. This demonstrates that metabolite detection windows and hence their added-value as target substance are unique and dependent on the analytical technique and not only on their pharmacokinetic behaviour. Consequently, in each case, a metabolite versus instrument evaluation is needed to maximise the probabilities of detecting doping offences.

New Insights into the Metabolism of Methyltestosterone and Metandienone: Detection of Novel A-Ring Reduced Metabolites.

Metandienone and methyltestosterone are orally active anabolic-androgenic steroids with a 17α-methyl structure that are prohibited in sports but are frequently detected in anti-doping analysis. Following the previously reported detection of long-term metabolites with a 17ξ-hydroxymethyl-17ξ-methyl-18-nor-5ξ-androst-13-en-3ξ-ol structure in the chlorinated metandienone analog dehydrochloromethyltestosterone ("oral turinabol"), in this study we investigated the formation of similar metabolites of metandienone and 17α-methyltestosterone with a rearranged D-ring and a fully reduced A-ring. Using a semi-targeted approach including the synthesis of reference compounds, two diastereomeric substances, viz. 17α-hydroxymethyl-17ß-methyl-18-nor-5ß-androst-13-en-3α-ol and its 5α-analog, were identified following an administration of methyltestosterone. In post-administration urines of metandienone, only the 5ß-metabolite was detected. Additionally, 3α,5ß-tetrahydro-epi-methyltestosterone was identified in the urines of both administrations besides the classical metabolites included in the screening procedures. Besides their applicability for anti-doping analysis, the results provide new insights into the metabolism of 17α-methyl steroids with respect to the order of reductions in the A-ring, the participation of different enzymes, and alterations to the D-ring.

An Effective Acid-Base-Induced Liquid-Liquid Microextraction Based on Deep Eutectic Solvents for Determination of Testosterone and Methyltestosterone in Milk.

An environmentally friendly method for the determination of testosterone and methyltestosterone by acid-base-induced deep eutectic solvents liquid-liquid microextraction (DES-ABLLME) combining with high-performance liquid chromatography was established. The deep eutectic solvent (DES) consisting of menthol:lauric acid:decanoic acid (3:1:1) can act as both hydrogen bond donor and hydrogen bond acceptor. In this approach, ammonia solution (NH3•H2O) is used as an emulsifier to react with DESs in the extraction process to generate salt and form milky white solution, achieving high extraction efficiency. Hydrochloric acid was used as a phase separator to change the emulsification state and promote the separation of extraction agent from water phase. A series of parameters were optimized including the volume of DES and the emulsifying agent, glucose concentration as well as hydrochloric acid volume. The method was linear in the range 0.5-100 µg mL-1 with a correlation coefficient (R) of 0.9999, and the limits of detection were 0.067 and 0.2 µg mL-1 for testosterone and methyltestosterone, respectively. This method was applied to analyze testosterone and methyltestosterone in milk samples, and the recoveries were between 89.2 and 108.2%.

Photodegradation of 17 α-methyltestosterone using TiO2 -Gd3+ and TiO2-Sm3+ photocatalysts and simulated solar radiation as an activation source.

According to the search in the state of the art, no antecedents were found in which photocatalytic degradation of 17α-methyltestosterone (MT) hormone has been carried out using doped-TiO2. Nor have the transformation products formed during the heterogeneous photocatalysis (FH) been identified. Therefore, in this study we analyzed the photocatalytic degradation of the MT in aqueous solution, using doped TiO2 with Sm3+ or Gd3+ at 0.3 and 0.5 %wt. Thermal treatment temperature (500 °C and 800 °C) and MT (20 mgL-1) mineralization were also studied. All photocatalysts were synthesized using the sol-gel method and characterized by X-ray Diffraction (XRD), Specific Surface Area (BET), Ultraviolet-visible Spectroscopy (UV-vis), High-Resolution Transmission Electron Microscope/Energy-Dispersive X-ray analysis (HRTEM/EDS) and, X-ray photoelectron spectroscopy (XPS), and photoluminescence (PL). MT mineralization was followed by a total organic carbon analyzer (TOC). The route of the photocatalytic mineralization of the hormone was obtained from the analysis of intermediate compounds determined by high performance liquid chromatography coupled to mass spectrometry (LC-TOF-MS). The results showed that TM and its transformation products were not degraded by photolysis. However, the degree of mineralization of the hormone was greater when the photocatalytic process was used. The photocatalytic efficiency was related to the dopant concentration, dopant type and thermal treatment. Therefore, Sm (0.3%)/TiO2 calcined at 500 °C showed the best performance for photocatalytic mineralization of MT.

Redox-Sensitive and Hyaluronic Acid-Functionalized Nanoparticles for Improving Breast Cancer Treatment by Cytoplasmic 17α-Methyltestosterone Delivery.

Novel reduction-responsive hyaluronic acid-chitosan-lipoic acid nanoparticles (HACSLA-NPs) were designed and synthesized for effective treatment of breast cancer by targeting Cluster of Differentiation 44 (CD44)-overexpressing cells and reduction-triggered 17α-Methyltestosterone (MT) release for systemic delivery. The effectiveness of these nanoparticles was investigated by different assays, including release rate, 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT), lactate dehydrogenase (LDH), caspase-3 activity, Rhodamine 123 (RH-123), and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). In vitro experiments revealed that Methyltestosterone/Hyaluronic acid-chitosan-lipoic acid nanoparticles (MT/HACSLA-NPs) illustrated a sustained drug release in the absence of glutathione (GSH), while the presence of GSH led to fast MT release. HACSLA-NPs also showed high cellular internalization via CD44 receptors, quick drug release inside the cells, and amended cytotoxicity against positive CD44 BT-20 breast cancer cell line as opposed to negative CD44, Michigan Cancer Foundation-7 (MCF-7) cell line. These findings supported that these novel reduction-responsive NPs can be promising candidates for efficient targeted delivery of therapeutics in cancer therapy.

Ameliorative effect of chitosan-conjugated 17α-methyltestosterone on testicular development in Clarias batrachus.

Chitosan nanoparticles conjugated with 17α-methyltestosterone (CS + MT) were used for studying their effect on the testicular development of Clarias batrachus during different reproductive phases. The size of chitosan nanoparticles was 127.2 nm and the nano-conjugated 17α methyltestosterone (17α-MT) was 196.1 nm (20 mg/100 ml of chitosan). Single injections of CS + MT at different doses such as 0.01, 0.1 and 0.5 µg/g body weight were administered to adults during the pre-spawning, spawning and post-spawning phase. Nano-conjugated steroid was effective at the lower dose; showing an increase in the Gonadosomatic Index (GSI) and 11-ketotestosterone level compared to the control group. Histological observations confirmed the dose-dependent advancement in spermatogenesis. These findings indicate the possibility of using CS + MT for enhancing gonadal maturity of C. batrachus.

Application of α- and ß-naphthoflavones as monooxygenase inhibitors of Absidia coerulea KCh 93, Syncephalastrum racemosum KCh 105 and Chaetomium sp. KCh 6651 in transformation of 17α-methyltestosterone.

In this work, 17α-methyltestosterone was effectively hydroxylated by Absidia coerulea KCh 93, Syncephalastrum racemosum KCh 105 and Chaetomium sp. KCh 6651. A. coerulea KCh 93 afforded 6ß-, 12ß-, 7α-, 11α-, 15α-hydroxy derivatives with 44%, 29%, 6%, 5% and 9% yields, respectively. S. racemosum KCh 105 afforded 7α-, 15α- and 11α-hydroxy derivatives with yields of 45%, 19% and 17%, respectively. Chaetomium sp. KCh 6651 afforded 15α-, 11α-, 7α-, 6ß-, 9α-, 14α-hydroxy and 6ß,14α-dihydroxy derivatives with yields of 31%, 20%, 16%, 7%, 5%, 7% and 4%, respectively. 14α-Hydroxy and 6ß,14α-dihydroxy derivatives were determined as new compounds. Effect of various sources of nitrogen and carbon in the media on biotransformations were tested, however did not affect the degree of substrate conversion or the composition of the products formed. The addition of α- or ß-naphthoflavones inhibited 17α-methyltestosterone hydroxylation but did not change the percentage composition of the resulting products.

Stability-Indicating TLC-Densitometric Assay for Methyltestosterone and Quantum Chemical Calculations.

Methyltestosterone is a synthetic testosterone derivative commonly used for the treatment of testosterone deficiency in males and one the anabolic steroids whose use is banned by World Anti-Doping Agency (WADA). This study presents a simple, cost-effective and rapid stability-indicating assay for densitometric quantification of methyltestosterone in pharmaceutical formulation. The developed method employed pre-coated TLC plates with mobile phase hexane:acetone (6.5:3.5 v/v). Limit of detection and limit of quantitation were found to be 2.06 and 6.24 ng/spot, respectively. Stress degradation study of methyltestosterone was conducted by applying various stress conditions such as hydrolysis under acidic, basic and neutral conditions, heating in anhydrous conditions and exposure to light. Methyltestosterone was found to be susceptible to photodegradation, acidic and basic hydrolysis. Degraded products were well resolved with significantly different Rf values. Acid degraded product was identified as 17,17-dimethyl-18-norandrosta-4,13(14)-dien-3-one through spectroscopic methods. The reactivity of methyltestosterone under applied stress conditions was also explained by quantum chemical calculations. The developed method is found to be repeatable, selective and accurate for quantification of methyltestosterone and can be employed for routine analysis.

Properties of PLA/PCL particles as vehicles for oral delivery of the androgen hormone 17α-methyltestosterone.

The aim of this study was to produce PLA (poly(lactic acid)) and PCL (polycaprolactone) oral carriers through the precipitation of the polymer solutions using supercritical CO2 as an antisolvent for the controlled release of the hydrophobic model drug 17α-methyltestosterone (MT). Such drug is a steroidal hormone used orally to develop and sustain primary and secondary male sex characteristics, e.g. for female Nile tilapia sex reversal in aquaculture. The influence of hormone, PLA and PCL concentrations on particle formation was analyzed, showing that high PCL concentrations produced particles with rougher surfaces and greater mean diameters. The incorporation efficiency of MT ranged from 20 to 51%, and its addition resulted in increases in particle mean diameter from 23 to 54 µm. Aggregation was observed for particles incorporating or not MT and high concentrations of MT led to the formation of more amorphous structures, changing the thermal behavior of the particles. The exposure of the PLA/PCL particles to pH conditions simulating gastrointestinal fish conditions showed that hormone release fraction at acidic pH ranged from 8 to 63% (over 2h), while in the basic pH the proportion released varied from 23 to 60% (over 10h), reaching levels adequate for the desired in vivo activity.

Synthesis and chemical reactions of the steroidal hormone 17α-methyltestosterone.

Structural modifications of natural products with complex structures like steroids require great synthetic effort. A review of literature is presented on the chemistry of the steroidal hormone 17α-methyltestosterone that is approved by Food and Drug Administration (FDA) in the United States as an androgen for estrogen-androgen hormone replacement therapy treatment. The analog also offers special possibilities for the prevention/treatment of hormone-sensitive cancers. The testosterone skeleton has important functionalities in the molecule that can act as a carbonyl component, an active methylene compound, α,ß-unsaturated enone and tertiary hydroxyl group in various chemical reactions to access stereoisomeric steroidal compounds with potent activity. In addition, microbiological methods of synthesis and transformation of this hormone are presented.

Tentative Structural Assignment of a Glucuronide Metabolite of Methyltestosterone in Tilapia Bile by Liquid Chromatography-Quadrupole-Time-of-Flight Mass Spectrometry.

Methyltestosterone (MT), a strong androgenic steroid, is not approved for use in fish aquaculture in the United States. It is used in the U.S. under an investigational new animal drug exemption (INAD) only during the early life stages of fish. There is a possibility that farmers feed fish with MT to enhance production for economic gains. Therefore, there is a need to develop methods for the detection of MT and its metabolite residues in fish tissue for monitoring purposes. Previously, our laboratory developed a liquid chromatography-quadrupole time-of-flight (LC-QTOF) method for characterization of 17-O-glucuronide metabolite (MT-glu) in bile of tilapia dosed with MT. The system used was an Agilent 6530 Q-TOF equipped with electrospray jet stream technology, operating in positive ion mode. Retrospective analysis of the data generated in that experiment by a feature-finding algorithm, combined with a search against an in-house library of possible MT-metabolites, resulted in the discovery of a major glucuronide metabolite of MT in the bile extracts. Preliminary data indicate it to be a glucuronide of a hydroxylated MT (OHMT-glu) which persists in tilapia bile for at least 2 weeks after dosing. We present the tentative structural assignment of the OHMT-glu in tilapia bile and time course of development. This glucuronide can serve as a marker to monitor illegal use of MT in tilapia culture.

Analysis of anabolic steroids in urine by gas chromatography-microchip atmospheric pressure photoionization-mass spectrometry with chlorobenzene as dopant.

A gas chromatography-microchip atmospheric pressure photoionization-tandem mass spectrometry (GC-µAPPI-MS/MS) method was developed for the analysis of anabolic androgenic steroids in urine as their trimethylsilyl derivatives. The method utilizes a heated nebulizer microchip in atmospheric pressure photoionization mode (µAPPI) with chlorobenzene as dopant, which provides high ionization efficiency by producing abundant radical cations with minimal fragmentation. The performance of GC-µAPPI-MS/MS was evaluated with respect to repeatability, linearity, linear range, and limit of detection (LOD). The results confirmed the potential of the method for doping control analysis of anabolic steroids. Repeatability (RSD<10%), linearity (R(2)≥0.996) and sensitivity (LODs 0.05-0.1ng/mL) were acceptable. Quantitative performance of the method was tested and compared with that of conventional GC-electron ionization-MS, and the results were in good agreement.

When color fails: illicit blue tablets containing anabolic androgen steroids.

The necessity of specific, confirmatory tests in the identification of seized illicit products was highlighted by the analysis of eighteen heart shaped, blue tablets confiscated by Police at a street control in the North East of Italy. The tablets responded as amphetamines to a preliminary color test (Marquis); a subsequent, confirmatory assay by gas chromatography-mass spectrometry revealed the presence of two anabolic androgen steroids (AAS), methandienone and methyltestosterone, in concentration of 1.7 and 1.5mg respectively per tablet; no trace of amphetamine-like or nitrogen containing compounds was found. The observed orange coloration was due to the reaction of concentrated sulphuric acid, contained in the Marquis reagent, with the Δ(4) C-3 keto group of steroids. The two AAS, banned under the world antidoping code, are not considered as psychoactive drugs of abuse in most countries, although their trafficking may entangle severe public health concerns.

Alternative long-term markers for the detection of methyltestosterone misuse.

Methyltestosterone (MT) is one of the most frequently detected anabolic androgenic steroids in doping control analysis. MT misuse is commonly detected by the identification of its two main metabolites excreted as glucuronide conjugates, 17α-methyl-5α-androstan-3α,17ß-diol and 17α-methyl-5ß-androstan-3α,17ß-diol. The detection of these metabolites is normally performed by gas chromatography-mass spectrometry, after previous hydrolysis with ß-glucuronidase enzymes, extraction and derivatization steps. The aim of the present work was to study the sulphate fraction of MT and to evaluate their potential to improve the detection of the misuse of the drug in sports. MT was administered to healthy volunteers and urine samples were collected up to 30days after administration. After an extraction with ethyl acetate, urine extracts were analysed by liquid chromatography tandem mass spectrometry using electrospray ionisation in negative mode by monitoring the transition m/z 385 to m/z 97. Three diol sulphate metabolites (S1, S2 and S3) were detected. Potential structures for these metabolites were proposed after solvolysis and mass spectrometric experiments: S1, 17α-methyl-5ß-androstan-3α,17ß-diol 3α-sulphate; S2, 17ß-methyl-5α-androstan-3α,17α-diol 3α-sulphate; and S3, 17ß-methyl-5ß-androstan-3α,17α-diol 3α-sulphate. Synthesis of reference compounds will be required in order to confirm the structures. The retrospectivity of these sulphate metabolites in the detection of MT misuse was compared with the obtained with previously described metabolites. Metabolite S2 was detected up to 21days after MT administration, improving between 2 and 3 times the retrospectivity of the detection compared to the last long-term metabolite of MT previously described, 17α-hydroxy-17ß-methylandrostan-4,6-dien-3-one.

Tandem mass spectrometry approach for the investigation of the steroidal metabolism: structure-fragmentation relationship (SFR) in anabolic steroids and their metabolites by ESI-MS/MS analysis.

Electrospray ionization tandem mass spectrometry (ESI-MS/MS) was used to investigate the effect of different substitutions introduced during metabolism on fragmentation patterns of four anabolic steroids including methyltestosterone, methandrostenolone, cis-androsterone and adrenosterone, along with their metabolites. Collision-induced dissociation (CID) analysis was performed to correlate the major product ions of 19 steroids with structural features. The analysis is done to portray metabolic alteration, such as incorporation or reduction of double bonds, hydroxylations, and/or oxidation of hydroxyl moieties to keto functional group on steroidal skeleton which leads to drastically changed product ion spectra from the respective classes of steroids, therefore, making them difficult to identify. The comparative ESI-MS/MS study also revealed some characteristic peaks to differentiate different steroidal metabolites and can be useful for the unambiguous identification of anabolic steroids in biological fluid. Moreover, LC-ESI-MS/MS analysis of fermented extract of methyltestosterone, obtained by Macrophomina phaseolina was also investigated.

Biotransformation of 17α-methyltestosterone in sediment under different electron acceptor conditions.

17α-Methyltestosterone (MT), an anabolic androgenic steroid, is used widely in inducing an all male population in aquaculture farming of fish, such as Nile tilapia (Oreochromis niloticus). Current understanding of the occurrence and fate of MT in the sediments and the surrounding areas of the aquaculture ponds are very limited. Bioassay tests showed that MT was biotransformed under aerobic and sulfate-reducing conditions with a half-life of 3.8d and 5.3d, respectively, with complete disappearance of androgenic activity. However, under methanogenic condition, MT was found to biotransform but the androgenic activity continued to persist even after 45 d of incubation. In contrast, MT was found to transform slowly under iron(III)-reducing condition and was hardly transformed under nitrate-reducing condition. A possible reason for the lack of transformation of MT under nitrate-reducing condition is the presence of the methyl group at the C-17 position. The results of this study suggest that MT and its degradation products with androgenic activity may potentially accumulate in the sediments of fish farming ponds under iron(III)-reducing, nitrate-reducing and methanogenic conditions.

[Component analysis of a new anabolic androgenic steroid and its monitoring research in human urine].

A new oral drug containing an unknown anabolic androgenic steroid (AAS) was studied. The principal constituent of the unknown anabolic hormone was studied by infrared spectrum (IR), nuclear magnetic resonance spectroscopy (1H NMR), ultraviolet spectroscopy (UV), and mass spectroscopy (MS). It was inferred to be methyl-1-testosterone (M1T, 17beta-hydroxy-17alpha-methyl-5alpha-androst-1-en-3-one) which was just added to the prohibited list in 2006. In addition, a monitoring, screening and confirmation of methyl-1-testosterone was established. The detection limit (S/N = 3) was 2 ng/mL, and the limit of quantification (S/N = 10) was 10 ng/mL. The relative standard deviation was 9.8% (n = 7) for the determination of pretreated urine sample with internal standard. This method was successfully applied in the identification of M1T positive urine. The excretion curve of M1T in human urine is described. It is a significant work for the discovery, determination and monitoring of the new AAS.

Determination of the hormonal growth promoter 17alpha-methyltestosterone in food-producing animals: bovine hair analysis by HPLC-MS/MS.

This paper describes the development, validation and application of a confirmatory method to detect 17alpha-methyltestosterone (MT) in bovine hair, to aid in controlling the administration of this growth promoter in meat-producing animals. After cryogenic grinding, MT was removed from the hair matrix using a single step extraction procedure with acetonitrile. Hydroxylamine derivatisation was used to enhance analyte determination with an electrospray ionisation (ESI) source. Determination was carried out using a triple quadrupole liquid chromatography tandem mass spectrometer (LC-MS/MS) in multiple reaction monitoring mode (MRM). The method was validated in accordance with the criteria defined in Commission Decision 2002/657/EC and using deuterated testosterone (T-d(3)) as the internal standard. The decision limit (CCalpha) was 0.07 ng g(-1) and the detection capability (CCbeta) was 0.12 ng g(-1). Repeatability was CV% (7%), within-laboratory reproducibility was CV% (11.0%), and trueness was (87%). Applicability of the method was demonstrated in an animal study. Samples obtained from animal experiments were analyzed and the presence of MT was confirmed.

Study on the molecularly imprinted polymers with methyl-testosterone as the template.

Molecularly imprinted polymers (MIPs) using methyl-testosterone as the template, methacrylic acid (MAA) as the monomer and ethylene glycol dimethacrylate (EDMA) as the crosslinker were prepared by precipitation polymerization. The morphology of the obtained particles was characterized by scanning electron microscopy (SEM) and the pore size was measured by BET. Then, the specificity and selectivity of the MIPs were evaluated using the equilibrium rebinding experiments. Besides, the MIPs were also used as the stationary phase of HPLC column and the retention behaviour to the template and analogues was confirmed using HPLC-MS-MS. Finally, the real application of the methyl-testosterone imprinted polymers was evaluated using SPE procedure with the spiked tap water and lake water. The results indicated that the prepared methyl-testosterone imprinted polymer showed specific rebinding ability to its template and could retain the template strongly compared with other structural analogues. At the same time, the MIPs could be used as SPE column to enrich methyl-testosterone in the lake water and show broad prospects in real samples.

Detection and characterization of a new metabolite of 17alpha-methyltestosterone.

The misuse of the anabolic steroid methyltestosterone is currently routinely monitored in doping control laboratories by gas chromatography-mass spectrometry (GC-MS) of two of its metabolites: 17alpha-methyl-5beta-androstane-3alpha,17beta-diol and 17alpha-methyl-5alpha-androstane-3alpha,17beta-diol. Because of the absence of any easy ionizable moiety, these metabolites are poorly detectable using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI). In this study, the metabolism of methyltestosterone has been reinvestigated by the use of a precursor ion scan method in LC-ESI-MS/MS. Two metabolites have been detected using this method. Both compounds have been confirmed in postadministration urine samples of an urokinase plasminogen activator-severe combined immunodeficiency (uPA-SCID) mouse with humanized liver and were characterized by LC-MS/MS and GC-MS using both quadrupole and time of flight analyzers. From the detailed study of the fragmentation, these metabolites were proposed to be epimethyltestosterone and a dehydrogenated compound. Epimethyltestosterone has previously been described as a minor metabolite, whereas the occurrence of the oxidized metabolite has not been reported. Comparison with the synthesized reference revealed that the structure of the dehydrogenated metabolite is 6-ene-epimethyltestosterone. A selected reaction monitoring method including three transitions for each metabolite has been developed and applied to samples from an excretion study and to samples declared positive after GC-MS analysis. 6-Ene-epimethyltestosterone was found in all samples, showing its applicability in the detection of methyltestosterone misuse.