Hydrogen Carrier Gas for GC/MS/MS Analysis of Steroids in Urine in Under 10 Minutes

Significance of the Topic

Reliable detection and quantification of endogenous anabolic–androgenic steroids in urine are critical for anti-doping laboratories to comply with World Anti-Doping Agency (WADA) requirements and uphold fair competition. The replacement of helium with hydrogen as a carrier gas addresses global helium shortages and escalating costs, offering a sustainable, cost-effective, and high-throughput analytical solution.

Objectives and Study Overview

This work aimed to develop and validate a rapid GC/MS/MS method using hydrogen carrier gas to quantify 14 endogenous anabolic steroids in urine within 10 minutes. The method targets a 40% reduction in run time compared to traditional helium-based protocols while meeting WADA limits of identification (LOIs) and demonstrating comparable analytical performance.

Methodology and Instrumentation

• Sample Preparation: Automated solid-phase extraction (SPE) on Bond Elut NEXUS cartridges, enzymatic hydrolysis, liquid–liquid extraction with methyl tert-butyl ether, and derivatization using MSTFA/NH4I/ethanethiol.
• Instrumentation: Agilent 7000C triple quadrupole GC/MS with Inert Plus EI source; hydrogen carrier gas at 1 mL/min; splitless injection of 1.5 µL; 12 m × 0.20 mm, 0.11 µm column; temperature program from 120 °C to 300 °C in 9.64 min.
• Calibration: Six-level, deuterated internal standard calibration curves with 1/x-weighted quadratic fits for each steroid.
• Data Processing: Agilent MassHunter acquisition and quantitative analysis software.

Main Results and Discussion

The hydrogen method achieved baseline separation of all steroid isomers in under 10 minutes, reducing analysis time by 40%. Calibration curves exhibited R² values above 0.997 across the required concentration ranges, meeting WADA MRPL and LOI criteria. No spectral changes were observed when using the standard Inert Plus EI source with hydrogen, and WADA external quality assessment (EQAS) samples produced z-scores below 1 for six analytes, confirming high accuracy and robustness.

Benefits and Practical Applications

• Throughput Improvement: Sub-10-minute runs increase daily sample capacity.
• Regulatory Compliance: Maintains sensitivity and selectivity required by WADA for endogenous steroids.
• Cost and Sustainability: Hydrogen is more abundant, less expensive, and can be produced via green electrolysis.
• Instrument Uptime: Hydrogen carrier gas keeps the EI source cleaner, reducing maintenance.

Future Trends and Opportunities

Broader adoption of hydrogen carrier gas in GC/MS workflows can extend to unknown-compound screening and analyses of hydrogen-reactive analytes using HydroInert sources. Coupling with automated high-throughput sample preparation and embracing sustainable laboratory practices will further enhance efficiency and environmental responsibility.

Conclusion

The validated hydrogen-based GC/MS/MS method delivers rapid, accurate, and robust quantification of endogenous anabolic steroids in urine, ensuring compliance with anti-doping standards while reducing analysis time and environmental impact.

References

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