In-vivo metabolic study of a new designer steroid substance 6β-chlorotestosterone

Code: 21C05GH

Objectives: 

The use of anabolic androgenic steroids (AAS) can increase body dimensions, muscular strength, and lean body mass in athletes. 6β-chloro-4-androsten-17β-ol-3-one (6β-chlorotestosterone), a new designer steroid substance, with an added 6-chloro group in the B-ring of a testosterone derivative, was reported existing in many dietary supplements [1]. This steroid substance is usually undetectable due to the lack of detection window in routine analysis [2]. Cheating athletes have a strong motive to take this designer steroid in order to both achieve performance enhancement and to escape from testing positive in anti-doping tests [3]. 

Methodology and experimental design:

1. Excretion studies will be conducted after 6β-chlorotestosterone took by 4 volunteers (two male, two female) at least. Urine samples will be collected and detected for 2 months or longer.
2. In order to evaluate the effect of 6β-chlorotestosterone to steroid profile, 6β-chlorotestosterone excretion urine samples will be analyzed by GC-MSn.
3. Steroid-like drugs are always extensive metabolized in human. Phase I metabolite and phase II metabolites will be extracted by different methods such as SPE or LLE. Enrichment procedure such as preparative performance liquid chromatography may also be employed for low concentration metabolites. 
4. Metabolites especially long term metabolites will be identified and characterized by LC-Q Extractive MS. Currently, liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) represents the most powerful metabolomics platform. Metabolites will be analyzed and identified in targeted mode and untargeted mode using accurate mass measurements.

Main findings

The aim of this study was to find urinary metabolic biomarkers of 6β-Cl-T for clinical research and doping control analysis. Additionally, the changes in the steroidal profile of human urine samples after administration of 6β-Cl-T were also evaluated GC-MS/MS. GC-C-IRMS was also employed to determine the endogenous and exogenous origins of T and its metabolites in samples exhibiting abnormally high T/E ratios following administration of 6β-Cl-T.

In the present study, a multi technique based analytical platform were utilized to identify 6β Cl T from black market and its in vivo epimeric metabolites, which included NMR, UPLC HRMS, HPLC MS/MS and GC MS/MS. Metabolic profile of 6β Cl T in human urine after an oral dose of 40 mg was reported for the first time.

Ten new metabolites including four phase I metabolites and six phase II metabolites were characterized and potentially identified. A solvolysis treatment after LC fraction procedure was employed to isolate and identify the ten metabolites, which was proven to be a powerful analytical tool for hunting down 6β Cl T metabolites. Two strategies

including 3α/β AAS conjugated metabolic mechanism and common rule of retention time variation between 5α/β AAS epimers were firstly introduced to distinguish 3α/β and 5α/β AAS epimeric metabolites. Elimination times of all new metabolites were investigated for the objective to improve and prolong the detection of 6β Cl T misuse, and the sulfate conjugate S1 was the potential long term metabolite detected up to 12 days by UPLC HRMS. Sulfate conjugates constituted a major metabolite class of 6β Cl T. Despite presenting mass spectral behavior referring to previous research experience, synthesis of the postulating metabolites should be conducted for unequivocal characterization of their

unequivocal characterization of their chemical structure. The influence on steroidal profile after administration of 6β-Cl-T was evaluated by GCwas evaluated by GC-MS/MS. Results showed t that 6β-Cl-T could transform to T in male volunteers, so as to cause an AAF for T and its metabolites. However there was no significant change in increasing testosterone levels for women individuals and UGT2B17 gene deletion (del/del) male individuals, which might cause false negative results in these people.