Extend the time window for the detection of low level anabolic‑androgenic steroids and their metabolites

Importance of the Topic

The detection and quantification of anabolic-androgenic steroids and their metabolites in urine are critical for effective doping control. Extending the detection window at low concentration levels enables anti-doping laboratories to identify illicit use of performance-enhancing substances long after administration. Advances in analytical instrumentation that improve sensitivity, robustness, and maintenance intervals directly impact compliance with World Anti-Doping Agency (WADA) requirements.

Objectives and Study Overview

This application note describes the use of the Agilent 7010 Triple Quadrupole GC/MS system for enhanced targeted screening and confirmation of anabolic steroids in urine samples. The study conducted at the Kreischa doping control laboratory in Germany aimed to achieve:

• Lower limits of detection for key steroid analytes and metabolites.
• A wider linear dynamic range covering both trace exogenous and abundant endogenous compounds.
• Compliance with WADA Minimum Required Performance Limits (MRPLs) for both screening and confirmation workflows.

Methodology and Instrumentation

The analytical setup consisted of an Agilent 7890 GC with multimode inlet, backflush capability, and an Agilent 7010 Triple Quadrupole MS. A 1 m precolumn and a 10 m × 0.18 mm × 0.18 µm analytical column with dimethyl polysiloxane film provided optimal chromatographic resolution and durability. Key parameters:

• Injection: 0.4 µL split 15:1 at 260 °C.
• Carrier gas: Helium with backflush at 16.5 min to remove high-boiling matrix components.
• Oven program: 130 °C hold, ramp to 186 °C at 56 °C/min, then to 206 °C at 2 °C/min, 221 °C at 5 °C/min, and final to 326 °C at 35 °C/min.
• MS conditions: Electron ionization with variable energy (70 eV and 130 eV time segments), source at 230 °C, quadrupoles at 150 °C, transfer line at 280 °C.

Main Results and Discussion

Optimal use of the 7010 TQ’s high sensitivity enabled injection of lower sample volumes while maintaining detection of low-abundance metabolites. By increasing electron energy to 130 eV during the elution window of highly abundant steroids (10.2–11.7 min), signal saturation was prevented and the linear dynamic range was extended fivefold compared to standard 70 eV operation. The method achieved:

• Detection limits down to 0.2 ng/mL for exogenous steroids.
• Quantitation of endogenous markers (androsterone, etiocholanolone) up to 1,000 ng/mL.
• Stable performance over >6,000 injections without ion source cleaning or column replacement, thanks to backflush removal of matrix residues.

Data review was performed using Agilent MassHunter Quantitative Analysis software for both quantitative single-point calibration of endogenous markers and qualitative review via Compounds-at-a-Glance.

Benefits and Practical Applications

The described protocol offers:

• Enhanced sensitivity for long-term metabolites, extending the detection window post administration.
• Robust operation and reduced maintenance through automated backflush.
• Simultaneous analysis of exogenous and endogenous steroids in a single run, streamlining workflow.

Future Trends and Potential Applications

Further developments may include:

• Integration of high-resolution mass spectrometry for broader untargeted screening.
• Automated data processing pipelines using machine learning for pattern recognition in steroid profiles.
• Miniaturized or field-deployable GC/MS systems for on-site screening at sporting events.

Conclusion

The Agilent 7010 GC/TQ system combined with optimized sample introduction, backflush technology, and dynamic electron energy control delivers a highly sensitive, robust, and WADA-compliant method for steroid profiling. This approach enhances the ability of doping control laboratories to detect low-level steroid use over extended periods with minimal maintenance.

References

1. D. Thieme et al. Drug Test. Anal. 2013, 5, 819.
2. WADA Technical Document TD2017MRPL, 2017.
3. WADA Technical Document TD2016EAAS, 2016.
4. J. Jakobsson et al. J. Clin. Endocrinol. Metab. 2006, 91, 687.