Detection of Oral-Turinabol Metabolites by LC/MS Q-TOF

Significance of the Topic

Effective detection of the synthetic steroid dehydrochloromethyltestosterone (Oral-Turinabol) is critical in anti-doping efforts to ensure fair competition and athlete health. Identifying reliable long-term biomarkers enhances the sensitivity and window of detection in doping control programs.

Objectives and Study Overview

This study applied high-resolution liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC/MS Q-TOF) to discover and characterize metabolites of Oral-Turinabol in human urine. Nine potential biomarkers were tentatively identified following administration of a single 5 mg oral dose to a healthy volunteer and weak basic liquid-liquid extraction without enzymatic hydrolysis.

Methodology and Instrumentation

Sample Preparation:

• Urine collection before dosing and up to 14 days post-dose, stored at –25 °C
• Liquid-liquid extraction at pH 9.5 using tert-butyl methyl ether
• Nitrogen evaporation and reconstitution in water:acetonitrile (9:1)

Chromatography:

• Agilent 1290 Infinite LC with Poroshell 120 EC-C18 column (2.1 × 50 mm, 2.7 µm)
• Gradient from 10% to 90% acetonitrile over 12 min, flow rate 0.4 mL/min, temperature 50 °C
• Injection volume of 20 µL

Mass Spectrometry:

• Agilent 6550 Accurate-Mass Q-TOF with iFunnel ESI source in negative ion mode
• Full-scan acquisition from 60 to 1100 m/z at 3 spectra/s with reference mass correction
• Targeted MS/MS for structural confirmation of metabolites

Results and Discussion

Nine metabolites (M1–M9) were detected and tentatively identified:

• M1–M4: glucuronide-conjugated species, involving hydroxylation and Wagner–Meerwein rearrangements
• M5–M9: unconjugated hydroxylated, dihydroxylated, trihydroxylated, and reduced metabolites

The predominant pathways include hydroxylation at positions 6, 12, or 16, glucuronidation, and molecular rearrangements. Metabolite M2, a rearranged glucuronide, exhibited the longest detection window, persisting beyond two weeks.

Benefits and Practical Applications

High-resolution LC/MS Q-TOF offers superior mass accuracy and the ability to detect phase II metabolites, significantly extending the detection window for Oral-Turinabol abuse. These findings enable anti-doping laboratories to refine and enhance routine screening protocols.

Future Trends and Potential Applications

Continued advances in high-resolution mass spectrometry and expanded metabolite libraries will improve structural elucidation and isomer differentiation. Integration of newly identified biomarkers into multi-analyte LC-triple quadrupole methods will further strengthen long-term detection in sports drug testing.

Conclusion

This work provides the first report of phase II metabolites of Oral-Turinabol using LC/MS Q-TOF. Metabolite M2 emerges as a promising long-term marker. Future efforts will focus on translating these markers into routine triple quadrupole workflows for enhanced doping control.

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