Alternative markers for Methylnortestosterone misuse in human urine

Area of Science:

• Forensic Science
• Analytical Chemistry
• Biochemistry

Background:

• Methylnortestosterone is a prohibited synthetic androgenic anabolic steroid.
• Current detection methods rely on identifying parent compounds and glucuronide metabolites.
• There is a need for improved analytical methods for detecting methylnortestosterone misuse.

Purpose of the Study:

• To directly detect and characterize methylnortestosterone sulfate-conjugated metabolites.
• To develop and validate new analytical approaches for identifying these metabolites.
• To enhance the detection capabilities for methylnortestosterone misuse in anti-doping efforts.

Main Methods:

• Direct detection of sulfate metabolites using LC/Q/TOF-MS in negative ionization mode after ethyl acetate extraction.
• Characterization of metabolites via GC-MS after solvolysis and TMS derivatization.
• Comparison of analytical data with synthesized reference standards of methylnortestosterone diol structures.

Main Results:

• Two main sulfate metabolites, S1 (17α-methyl-5α-estrane-3α, 17β-diol 3α sulfate) and S2 (17α-methyl-5β-estrane-3α, 17β-diol 3α sulfate), were detected.
• S1 was detected up to 72 hours and S2 up to 192 hours post-administration.
• Additional glucuronide (M3, M4) and sulfate (S3) metabolites were identified, with S2 also detected as a GC-MS/MS artifact.

Conclusions:

• Direct analysis of methylnortestosterone sulfate metabolites is feasible and effective.
• The identified sulfate metabolites (S1, S2, S3) provide new targets for anti-doping detection.
• These findings significantly advance the analytical methods for combating methylnortestosterone doping.