Searching for Alternative Danazol Metabolites Through Accurate Mass with LC/MS Q-TOF

Significance of the Topic

The detection of subtle steroid metabolites is essential for robust antidoping efforts. High-resolution LC/MS Q-TOF allows accurate-mass measurements that facilitate the discovery of previously unreported markers in urine samples. This capability enhances the retrospective detection of prohibited compounds such as danazol.

Objectives and Study Overview

This study aimed to identify alternative urinary markers of danazol metabolism using an LC/MS Q-TOF platform. An excretion study was performed following a single 100 mg oral dose of danazol in a healthy male volunteer, with urine samples collected up to 30 days post-administration. The focus was on detecting mono- and dihydroxylated derivatives beyond the known metabolites ethisterone (M1) and 2α-hydroxymethylethisterone (M2).

Methodology and Instrumentation

A simple liquid–liquid extraction protocol was applied to 6 mL urine aliquots under basic (carbonate buffer pH 11, TBME) and acidic (formic acid pH 2, ethyl acetate) conditions. Extracts were evaporated and reconstituted in 150 µL of mobile phase.

• Chromatography: Agilent Poroshell 120 EC-C18 column (2.1×50 mm, 2.7 µm) with gradient elution (10–75% acetonitrile over 12 min; up to 90% in 12.1 min), flow rate 0.4 mL/min, 50 °C.
• Mass Spectrometry: Agilent 6550 Accurate-Mass Q-TOF with Jet Stream ESI source operated in positive and negative modes. Full-scan acquisition (m/z 60–1100) at 3 spectra/s; targeted MS/MS at 1.5 spectra/s with 35 eV collision energy.
• Data Processing: Agilent MassHunter Workstation software for data acquisition and qualitative analysis.

Key Results and Discussion

More than 20 danazol-related metabolites were detected by comparing post-dose and baseline samples. Notably, four novel dihydroxylated species—two isomers each of danazol PC+2OH and danazol M1+2OH—were identified. MS/MS fragmentation patterns supported the structural assignments. The most persistent marker, one isomer of danazol M2+2OH, remained detectable up to 16 days after dosing, significantly extending the monitoring window compared to traditional markers.

Benefits and Practical Applications

The discovery of new hydroxylated metabolites offers enhanced sensitivity and longer retrospective detection periods in doping control protocols. Implementation of these markers in routine screening can improve the identification of danazol misuse in athletes.

Future Trends and Potential Applications

Advances in high-resolution, accurate-mass spectrometry and non-targeted data-mining approaches will likely reveal additional metabolite patterns for various prohibited substances. Integration of automated MS/MS spectral interpretation and robust databases will further streamline anti-doping analyses.

Conclusion

Accurate-mass LC/MS Q-TOF analysis enabled the identification of novel dihydroxylated danazol metabolites, broadening the arsenal of biomarkers available for doping control. These findings support the adoption of high-resolution workflows to improve detection capabilities and retrospective monitoring of steroid misuse.

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