Analysis of Anabolic Steroids for Doping Control Purposes by GC-TOFMS and GCxGC-TOFMS

Significance of the Topic

Anabolic steroids are among the most commonly abused substances in sports, and their reliable detection at trace levels is essential for fair competition and athlete health. Traditional one-dimensional GC-MS methods rely on selective ion monitoring or MS/MS and may overlook novel or unexpected analogues. Comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GCxGC-TOFMS) offers full-scan capability and enhanced separation power, improving sensitivity, confirmation reliability, and non-target screening potential.

Objectives and Study Overview

This study evaluated and compared the performance of one-dimensional GC-TOFMS and GCxGC-TOFMS for the analysis of five steroidal doping agents at the low concentration required by WADA (2 ng/mL in urine). The goals were to determine limits of detection, signal-to-noise ratios, mass spectral match factors, and the overall suitability of each approach for screening and confirmatory analysis in a complex biological matrix.

Methodology and Instrumentation

Mixed standards of clenbuterol, 19-norandrosterone, epimethendiol, M2-metabolite of methyltestosterone, and 3′OH-stanozolol were prepared. Urine extracts were spiked to 2 ng/mL and derivatized to trimethylsilyl (TMS) derivatives. Methyltestosterone served as an internal standard at 50 ng/mL.

Instrumentation

• GC-TOFMS: Agilent 7890N GC with BP-5 column (30 m × 0.25 mm × 0.25 µm), LECO Pegasus HT TOFMS, EI –70 eV, acquisition at 4 spectra/s.
• GCxGC-TOFMS: LECO Pegasus 4D on the same GC, thermal loop modulator, secondary oven, BPX-50 second column (2.5 m × 0.1 mm × 0.1 µm), helium carrier at 1 mL/min, modulation period 3 s, acquisition at 200 spectra/s.

Key Results and Discussion

One-dimensional GC-TOFMS achieved signal-to-noise ratios of 77–299 for primary ions and match factors >670 for most analytes at 2 ng/mL, except for epimethendiol which was poorly detected. In contrast, GCxGC-TOFMS delivered up to tenfold improvement in signal-to-noise, with all five steroids detected at 2 ng/mL, match factors typically >900 (863 for epimethendiol), and estimated sub-pg LODs.

The enhanced chromatographic resolution of GCxGC reduced matrix interferences, allowing clear mass spectra and reliable identification based on full-scan data. Automated deconvolution and library matching in ChromaTOF facilitated rapid processing and non-targeted discovery.

Benefits and Practical Applications

• Improved sensitivity and confirmation at ultra-trace levels support WADA requirements for screening and confirmatory tests.
• Full mass spectral acquisition enables retrospective analysis and identification of emerging or unknown steroids.
• Automated deconvolution and spectral library searching streamline data processing in doping control labs.

Future Trends and Applications

Advances in high-resolution TOFMS, improved modulators, and data analytics will further enhance non-target screening and quantitation. Integration with machine learning and comprehensive spectral libraries will accelerate the identification of novel performance-enhancing compounds. Miniaturized systems and faster GCxGC methods could increase sample throughput and accessibility in routine anti-doping workflows.

Conclusion

GCxGC-TOFMS outperforms conventional one-dimensional GC-TOFMS for the analysis of anabolic steroids in urine, offering superior sensitivity, spectral quality, and the ability to detect unexpected compounds. Its implementation in doping control laboratories can strengthen both screening and confirmatory capabilities.

References

• Mitrevski et al.: Application of comprehensive two-dimensional gas chromatography to sterols analysis, J. Chromatogr. A 1214 (2008) 134–142.
• Mitrevski et al.: Evaluation of World Anti-Doping Agency criteria for anabolic agent analysis by using comprehensive two-dimensional gas chromatography–mass spectrometry, Anal. Bioanal. Chem. 396 (2010) 2503–2511.