Glomerella fusarioides-catalyzed structural transformation of steroidal drugs mesterolone and methasterone, and anti-inflammatory activity of resulting derivatives.

Transformation of steroidal drug mesterolone (1) with Glomerella fusarioides yielded two new (17α-hydroxy-1α-methyl-5α-androstan-3-one-11α-yl acetate (2) and 15α-hydroxy-1-methyl-5α-androstan-1-en-3,17-dione (3)), and four known derivatives (15α,17ß-dihydroxy-1α-methyl-5α-androstan-3-one (4), 15α-hydroxy-1α-methyl-5α-androstan-3,17-dione (5), 1α-methyl-androsta-4-en-3,17-dione (6) and 15α,17ß-dihydroxy-1-methyl-5α-androstan-1-en-3-one (7). Similarly, G. fusarioides-catalyzed transformation of steroidal drug methasterone (8) afforded four new metabolites, 11α,17ß-dihydroxy-2,17α-dimethylandrosta-1,4-diene-3-one (9), 3a,11α,17ß-trihydroxy-2α,17α-dimethyl-5α-androstane (10), 1ß,3ß,17ß-trihydroxy-2α,17α-dimethyl-5α-androstane (11), and 11α,17ß-dihydroxy-2,17α-dimethylandrosta-1,4-diene-3-one (12). Structures of new derivatives were determined by using 1D-, and 2D-NMR, HREI-MS, and IR spectroscopic data. New derivative 3 was identified as a potent inhibitor of NÈ® production with the IC50 value of 29.9 ± 1.8 µM, in comparison to the standard l-NMMA (IC50 = 128.2 ± 0.8 µM) in vitro. In addition, methasterone (8) (IC50 = 83.6 ± 0.22 µM) also showed a significant activity comparable to new derivative 12 (IC50 = 89.8 ± 1.2 µM). New derivatives 2 (IC50 = 102.7 ± 0.5 µM), 9 (IC50 = 99.6 ± 5.7 µM), 10 (IC50 = 123.5 ± 5.7 µM), and 11 (IC50 = 170.5 ± 5.0 µM) showed a moderate activity. NG-MonomethylL-arginine acetate (IC50 = 128.2 ± 0.8 µM) was used as standared NO⋅- free radicals have an important role in the regulation of immune responses and cellular events. Their overproduction is associated with the pathogenesis of numerous ailments, such as Alzheimer's cardiac disorders, cancer, diabetes, and degenerative diseases. Therefore, inhibition of NÈ® production can help in the treatment of chronic inflammation and associated disorders. All derivatives were found to be non-cytotoxic to human fibroblast (BJ) cell line. The results presented here form the basis of further research for the development of new anti-inflammatory agents with improved efficacy through biotransformation approaches.

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.

Gas chromatography-mass spectrometry analysis of non-hydrolyzed sulfated steroids by degradation product formation.

Steroid detection and identification remain key issues in toxicology, drug testing, medical diagnostics, food safety control, and doping control. In this study, we evaluate the capabilities and usefulness of analyzing non-hydrolyzed sulfated steroids with gas chromatography-mass spectrometry (GC-MS) instead of the conventionally applied liquid chromatography-mass spectrometry (LC-MS) approach. Sulfates of 31 steroids were synthesized and their MS and chromatographic behavior studied by chemical ionization-GC-triple quadrupole MS (CI-GC-TQMS) and low energy-electron ionization-GC-quadrupole time-of-flight-MS (LE-EI-GC-QTOF-MS). The collected data shows that the sulfate group is cleaved off in the injection port of the GC-MS, forming two isomers. In CI, the dominant species (ie, [MH - H2 SO4 ]+ or [MH - H4 S2 O8 ]+ for bis-sulfates) is very abundant due to the limited amount of fragmentation, making it an ideal precursor ion for MS/MS. In LE-EI, [M - H2 SO4 ].+ and/or [M - H2 SO4 - CH3 ].+ are the dominant species in most cases. Based on the common GC-MS behavior of non-hydrolyzed sulfated steroids, two applications were evaluated and compared with the conventionally applied LC-MS approach; (a) discovery of (new) sulfated steroid metabolites of mesterolone and (b) expanding anabolic androgenic steroid abuse detection windows. GC-MS and LC-MS analysis of non-hydrolyzed sulfated steroids offered comparable sensitivities, superseding these of GC-MS after hydrolysis. For non-hydrolyzed sulfated steroids, GC-MS offers a higher structural elucidating power and a more straightforward inclusion in screening methods than LC-MS.

Identification and characterization of novel long-term metabolites of oxymesterone and mesterolone in human urine by application of selected reaction monitoring GC-CI-MS/MS.

The search for metabolites with longer detection times remains an important task in, for example, toxicology and doping control. The impact of these long-term metabolites is highlighted by the high number of positive cases after reanalysis of samples that were stored for several years, e.g. samples of previous Olympic Games. A substantial number of previously alleged negative samples have now been declared positive due to the detection of various long-term steroid metabolites the existence of which was unknown during the Olympic Games of 2008 and 2012. In this work, the metabolism of oxymesterone and mesterolone, two anabolic androgenic steroids (AAS), was investigated by application of a selected reaction monitoring gas chromatography-chemical ionization-triple quadrupole mass spectrometry (GC-CI-MS/MS) protocol for metabolite detection and identification. Correlations between AAS structure and GC-CI-MS/MS fragmentation behaviour enabled the search for previously unknown but expected AAS metabolites by selection of theoretical transitions for expected metabolites. Use of different hydrolysis protocols allowed for evaluation of the detection window of both phase I and phase II metabolites. For oxymesterone, a new metabolite, 18-nor-17ß-hydroxymethyl-17α-methyl-4-hydroxy-androst-4,13-diene-3-one, was identified. It was detectable up to 46 days by using GC-CI-MS/MS, whereas with a traditional screening (detection of metabolite 17-epioxymesterone with electron ionization GC-MS/MS) oxymesterone administration was only detectable for 3.5 days. A new metabolite was also found for mesterolone. It was identified as 1α-methyl-5α-androstan-3,6,16-triol-17-one and its sulfate form after hydrolysis with Helix pomatia resulted in a prolonged detection time (up to 15 days) for mesterolone abuse. Copyright © 2017 John Wiley & Sons, Ltd.

Markers of mesterolone abuse in sulfate fraction for doping control in human urine.

This manuscript describes the direct detection of mesteroloe sulfo-conjugated metabolites by liquid chromatography/quadrupole/time of flight mass spectrometry (LC/Q/TOFMS) with special focus on evaluation of their retrospective detectability and their structure elucidation. A comparison of their long-term detectability, with the mesterolone main metabolite (1α-methyl-5α-androstan-3α-ol-17-one) excreted in glucuronide fraction and detected by gas chromatography/high resolution mass spectrometry (GC/HRMS), is also presented. Studies on mesterolone were performed with samples obtained from two excretion studies after single oral administration of Proviron© by healthy volunteers. Potential sulfate metabolites were detected in post administration samples after liquid-liquid extraction (LLE) with ethyl acetate and LC/TOFMS analysis, in negative mode. Twelve mesterolone sulfate metabolites from the first excretion study and nine from the second were subsequently confirmed by LC/Q/TOFMS. Finally, six mesterolone sulfate metabolites were considered important taking into account their abundance and long-term detectability, encoded as M1, M2, M4, M5, M6 and M7. The proposed mesterolone sulfate metabolites M1, M2, M4 and M5 (excreted as sulfates) have the same retrospectivity with the main mesterolone metabolite, excreted in glucuronide fraction. For metabolite characterization, LC fractionation was performed. The metabolites were identified and characterized by GC/MS, after solvolysis and derivatization. Combined mass spectra data from trimethyl-silyl (TMS), TMS-enolTMS and methoxime-TMS derivatives were taken into account for the characterization of these metabolites. It was concluded that M1 is 1α-methyl-5α-androstan-3ß-ol-17 one, M2 is 1α-methyl-5α-androstan-3α-ol-17 one, M4 is 1α-methyl-5a-androstan-3ß, 16z-diol-17-one, M5 is 1α-methyl-5α-androstan-17z,4ξ-diol-3one, M6 is 1α-methyl-5α-androstan-3z,6z-diol-17-one and M7 is 4z-hydroxy-1α-methyl-5α-androstan-3,17-dione.

New potential biomarkers for mesterolone misuse in human urine by liquid chromatography quadrupole time-of-flight mass spectrometry.

In this paper, mesterolone metabolic profiles were investigated carefully. Mesterolone was administered to one healthy male volunteer. Urinary extracts were analyzed by liquid chromatography quadruple time-of-flight mass spectrometry (LC-QTOFMS) for the first time. Liquid-liquid extraction was applied to processing urine samples, and dilute-shoot analyses of intact metabolites were also presented. In LC-QTOFMS analysis, chromatographic peaks for potential metabolites were hunt down by using the theoretical [M-H](-) as target ions in full scan experiment, and their actual deprotonated ions were analyzed in targeted MS/MS mode. Ten metabolites including seven new sulfate and three glucuronide conjugates were found for mesterolone. Because of no useful fragment ion for structural elucidation, gas chromatography-mass spectrometry instrumentation was employed to obtain structural details of the trimethylsilylated phase I metabolite released after solvolysis. Thus, their potential structures were proposed particularly by a combined MS approach. All the metabolites were also evaluated in terms of how long they could be detected, and S1 (1α-methyl-5α-androst-3-one-17ß-sulfate) together with S2 (1α-methyl-5α-androst-17-one-3ß-sulfate) was detected up to 9 days after oral administration, which could be the new potential biomarkers for mesterolone misuse.

Biotransformation of androgenic steroid mesterolone with Cunninghamella blakesleeana and Macrophomina phaseolina.

Fermentation of mesterolone (1) with Cunninghamella blakesleeana yielded four new metabolites, 1α-methyl-1ß,11ß,17ß-trihydroxy-5α-androstan-3-one (2), 1α-methyl-7α,11ß,17ß-trihydroxy-5α-androstan-3-one (3), 1α-methyl-1ß,6α,17ß-trihydroxy-5α-androstan-3-one (4) and 1α-methyl-1ß,11α,17ß-trihydroxy-5α-androstan-3-one (5), along with three known metabolites, 1α-methyl-11α,17ß-dihydroxy-5α-androstan-3-one (6), 1α-methyl-6α,17ß-dihydroxy-5α-androstan-3-one (7) and 1α-methyl-7α,17ß-dihydroxy-5α-androstan-3-one (8). Biotransformation of 1 with Macrophomina phaseolina also yielded a new metabolite, 1α-methyl, 17ß-hydroxy-5α-androstan-3,6-dione (9). The isolated metabolites were subjected to various in vitro biological assays, such as anti-cancer, inhibition of α-glucosidase, and phosphodiesterase-5 enzymes and oxidative brust. However, no significant results were observed. This is the first report of biotransformation of 1 with C. blakesleeana and M. phaseolina.