The Determination of Anabolic Steroids in Human Urine using the TSQ Quantum XLS

Importance of the Topic

Use of anabolic steroids in sports remains a critical concern for anti-doping authorities. Analytical methods capable of detecting and quantifying trace levels of performance-enhancing drugs in complex biological matrices are essential to ensure fair competition and compliance with regulatory guidelines.

Objectives and Study Overview

This application note describes the development and validation of a gas chromatography–tandem mass spectrometry (GC-MS/MS) method using the Thermo Scientific TSQ Quantum XLS instrument. The aim was to identify and quantify five anabolic steroids in human urine at low nanogram-per-milliliter concentrations while meeting World Anti-Doping Agency (WADA) criteria.

Methodology and Instrumentation

Sample introduction was automated using an AS 3000 II autosampler and TRACE GC Ultra equipped with a backflush programmable temperature vaporization (PTV) inlet. Chromatographic separation employed a 15 m TraceGOLD TG-5MS capillary column with helium carrier gas at 1.4 mL/min. Key GC conditions included a multi-segment oven program from 140 °C to 300 °C and a 2 µL splitless injection. The TSQ Quantum XLS operated in electron impact mode (70 eV) with a closed ion source at 280 °C, argon collision gas (1.5 mTorr), and 0.7 Da resolution on both quadrupoles. Selected reaction monitoring (SRM) was configured with two transitions per analyte plus two for a deuterated testosterone internal standard. Quantitation and confirmation were automated via QuanLab Forms software.

Main Results and Discussion

The method successfully detected clenbuterol, 19-norandrosterone, 17β-methyl-5β-androst-1-ene-3α,17α-diol, 17α-methyl-5β-androstane-3α,17β-diol, and 3′-hydroxystanozolol in five urine specimens. Calculated concentrations ranged from 0.42 to 23.6 ng/mL, each above the WADA limits of reporting (1–2 ng/mL). Ion ratio measurements remained within ±10–50% of base-peak criteria with relative standard deviations below 8%, demonstrating compliance with WADA identification thresholds. A comparison of early and late injections for the low-intensity 3′-hydroxystanozolol transition confirmed stable peak shape and signal intensity across 30 back-to-back analyses. PTV backflush removed matrix interferences, preserving column and source lifetime and ensuring consistent chromatographic performance.

Benefits and Practical Application of the Method

• High sensitivity and selectivity for multi-analyte detection at sub-nanogram levels
• Robust operation in complex urine matrices with minimal interference
• Automated data processing for streamlined confirmation and reporting
• Enhanced instrument uptime through PTV backflush matrix removal
• Efficient workflow supporting high sample throughput

Future Trends and Potential Applications

Advances may include coupling GC-MS/MS with rapid or multidimensional separations for broader analyte panels, integrating artificial intelligence for automated data review, miniaturization of instruments for field testing, and expansion of screening protocols to cover emerging designer steroids.

Conclusion

The TSQ Quantum XLS GC-MS/MS method with PTV backflush and SRM provides a sensitive, reproducible, and WADA-compliant approach for anabolic steroid analysis in urine. It delivers reliable identification and quantitation at low concentration levels, streamlines laboratory workflows, and minimizes maintenance requirements.

Reference

• World Anti-Doping Agency Technical Document TD2010IDCR, effective September 1, 2010.
• Munari F., Pelagatti S., Cadoppi A. Trace determination of organophosphorous pesticides in olive oil by GC analysis through PTV backflush/FPD; Application Note 10049; Thermo Fisher Scientific: Milan, Italy, 2004.