Detection of Stanozolol Glucuronides in Human Sports Drug Testing by Meansof High-Resolution, Accurate-MassMass Spectrometry

Significance of the Topic

Stanozolol is a widely abused anabolic steroid with challenging physicochemical properties that complicate its detection by classical GC-MS methods. Reliable identification of its glucuronide metabolites in urine is essential for effective anti-doping control and athlete safety.

Study Objectives and Overview

This study aimed to establish and validate a direct dilute-and-shoot LC-HRAM-MS approach for the unambiguous detection of stanozolol glucuronides in human urine. Key goals included:

• Characterizing 3ʼ-OH-stanozolol-O-glucuronide using commercially available reference material.
• Developing a rapid screening method and a confirmatory SPE-LC-HRAM-MS assay.
• Evaluating new long-term metabolites and monitoring excretion profiles up to 28 days post-administration.

Methodology and Instrumentation

Healthy male volunteers received a single 5 mg oral dose of stanozolol. Urine was sampled before and up to 28 days post-dose. Two sample preparation workflows were applied:

• Dilute-and-shoot: 90 µL urine spiked with methyltestosterone internal standard, diluted with acetonitrile, vortexed, and injected.
• Confirmatory SPE: 1 mL urine loaded on conditioned cartridge, washed, eluted with methanol, evaporated, and reconstituted.

Chromatography and mass spectrometry conditions:

• LC: Thermo Accela 1250 with Nucleodur C18 Pyramid column (50 × 2 mm, 1.8 µm), mobile phases 0.1% formic acid and acetonitrile, 200 µL/min gradient.
• MS: Thermo Q Exactive Focus with HESI-II source in positive ESI, Full Scan (m/z 100–1000) and targeted PRM of m/z 505.25 and 521.25, resolution 35,000 (FWHM), HCD at 55–72 eV.

Main Results and Discussion

High-resolution accurate-mass data allowed differentiation of stanozolol O- and N-glucuronides by retention times, enzymatic hydrolysis, and collision energy requirements. Key findings:

• Identification of two isomeric glucuronides: hydrolysable 17-O-glucuronide and N-glucuronide.
• Detection of 16-oxo-stanozolol glucuronide based on 30 Da loss of C2H6 and comparison with reference standard.
• Excretion profiling showed that 17-epistanozolol N-glucuronide remained detectable up to 28 days post-dose.
• Validation of the screening assay yielded an LOD of 20 pg/mL, acceptable specificity, ion suppression within 45%, and recovery around 106%.
• Confirmatory method demonstrated linearity (25–250 pg/mL, r²=0.994) and intraday/interday precision below 16% CV.

Benefits and Practical Applications

The developed LC-HRAM-MS workflow offers:

• Rapid sample preparation via dilute-and-shoot, reducing turnaround times.
• Enhanced sensitivity and specificity for stanozolol metabolite detection.
• Extended detection windows through novel long-term metabolites.
• Robust confirmation using SPE and high-resolution tandem MS.

Future Trends and Applications

Advances likely include:

• Isotopic labeling studies to further elucidate fragmentation pathways and conjugation sites.
• Expansion of HRAM-MS panels to cover emerging steroid metabolites.
• Automation of dilute-and-shoot workflows for high-throughput doping control labs.
• Integration with data‐driven screening tools for improved anti-doping strategies.

Conclusion

A combined dilute-and-shoot and confirmatory SPE‐LC-HRAM-MS approach was established and validated for stanozolol glucuronides, enabling confident detection of misuse and identification of long-term metabolites up to 28 days after administration.

Reference

1. Schänzer W., Guddat S., Thomas A., Opfermann G., Geyer H., Thevis M. Expanding analytical possibilities concerning the detection of stanozolol misuse by high resolution/high accuracy mass spectrometric detection of stanozolol glucuronides in human sports drug testing. Drug Test Anal. 2013 Nov-Dec;5(11-12):810-818.
2. Mareck U., Geyer H., Opfermann G., Thevis M., Schänzer W. Factors Influencing the Steroid Profile in Doping Control Analysis. J. Mass Spectrom. 2008;43:877-891.