Enhanced Detection and Separation of Anabolic Steroids for Anti-Doping Control Screening by Comprehensive Two- Dimensional Gas Chromatography Time-of-Flight Mass Spectrometry (GCxGC-TOFMS)

Importance of the Topic

The reliable detection of anabolic steroids in biological samples is essential for anti-doping agencies to uphold fair play and protect athlete health. Traditional one-dimensional gas chromatography often faces challenges resolving complex steroid mixtures in urine, leading to potential false negatives and legal risks. Comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GCxGC-TOFMS) offers enhanced separation power and rapid spectral acquisition, enabling more definitive identification and quantification of low-abundance steroids.

Aims and Study Overview

This study evaluates the capabilities of GCxGC-TOFMS for screening five androgenic anabolic steroids in urine at sub-part-per-billion concentrations. By comparing analytical performance against conventional methods, the research aims to demonstrate improved chromatographic resolution, sensitivity, and quantitative linearity in compliance with World Anti-Doping Agency (WADA) criteria.

Methodology and Instrumentation

Sample Preparation:

• Acid hydrolysis with β-glucuronidase to release conjugated steroids
• Liquid–liquid extraction using methyl-tert-butyl ether
• Nitrogen evaporation to dryness
• Trimethylsilyl derivatization using MSTFA reagent

Used Instrumentation

• LECO Pegasus 4D GCxGC-TOFMS system
• Agilent 7890 GC with two-stage cryogenic modulator and secondary oven
• Primary column: 30 m Rxi-5ms, 0.25 mm × 0.25 µm
• Secondary column: 1.2 m BPX-50, 0.18 mm × 0.20 µm
• Helium carrier gas at 1.5 mL/min constant flow
• ChromaTOF software for data processing and deconvolution

Main Results and Discussion

• Successful detection and identification of five steroids at 2 ng/mL (2 ppb) in urine, meeting WADA minimum performance levels.
• Signal-to-noise ratios ranged from 85 to 1353 at the 2 ppb level, exceeding required confirmation thresholds.
• Calibration curves displayed linearity above 99.9% over a 2–100 ng/mL range, enabling accurate quantification.
• GCxGC provided baseline resolution of coeluting metabolites, while TOFMS deconvolution algorithms isolated target spectra even under heavy matrix interferences.
• Limits of detection were calculated in the low picogram-per-milliliter range, demonstrating sub-ppt sensitivity.

Benefits and Practical Applications

The GCxGC-TOFMS approach enhances anti-doping workflows by providing deeper chromatographic separation, faster scan rates, and improved deconvolution capabilities. These advances reduce false negatives, streamline screening protocols, and support legal defensibility of analytical results in sports and regulatory laboratories.

Future Trends and Opportunities

• Integration with high-resolution mass analyzers for increased compound specificity.
• Automation of sample preparation to improve throughput and reproducibility.
• Expansion to other classes of performance-enhancing drugs and metabolites.
• Application of machine learning for pattern recognition in complex data sets.
• Miniaturized and field-deployable GCxGC platforms for on-site testing.

Conclusion

This work demonstrates that GCxGC-TOFMS significantly outperforms conventional one-dimensional GC-MS for anabolic steroid screening in urine. By delivering enhanced resolution, sub-part-per-billion sensitivity, and robust quantitative performance, the method meets and exceeds WADA requirements, offering a powerful tool for anti-doping control.