Detection of synthetic forms of endogenous anabolic androgenic steroids by GC-C-IRMS

Significance of Topic

The accurate detection of synthetic forms of endogenous anabolic androgenic steroids (EAAS) is critical in anti-doping efforts. Compound-specific stable carbon isotope analysis by GC-C-IRMS enables differentiation between naturally produced hormones and those derived from plant precursors based on their distinct δ13C signatures. This methodological approach underpins WADA regulations and safeguards fair competition in sports.

Objectives and Overview

This application note presents a complete GC-C-IRMS workflow compliant with WADA Technical Document TD2016IRMS for the analysis of testosterone (T) and key metabolites in urine. It highlights the integration of Agilent GC separation, microcombustion interface, visION isotope analyzer, and IONOS data software to achieve robust, high-throughput isotope ratio measurements.

Methodology and Used Instrumentation

Sample Preparation and Extraction

• Hydrolysis and SPE or HPLC purification to isolate T, 5α-adiol, 5β-adiol, epitestosterone (E), androsterone (A), etiocholanolone (Et), DHEA, 16-enol, pregnanediol (Pgdiol) and androstenol.
• Avoidance of derivatization eliminates additional carbon and simplifies mass balance corrections.

Chromatography and Combustion

• Agilent 7890B GC with capillary column and helium carrier gas for compound separation.
• GC-5 furnace interface ensures quantitative combustion to CO2 via microbore reactor with automated O2 bleed and recharge.

Isotope Analysis

• visION IRMS for automated ion source tuning, linearity checks and isotopic ratio determination.
• IONOS data software for integrated peak mapping, multi-level calibration, and automated calculation of ERC vs. target compound δ13C differences.

Main Results and Discussion

Precision and Sensitivity

• Negative and positive control samples yielded long-term δ13C precision ≤0.5‰.
• Reference standards exhibited standard deviations ≤0.2‰ over months of analysis.

Linearity and Limit of Quantification

• Instrument response linear between 0.1 and 17 nA peak heights; LOiQ set at 0.5 nA (~4 ng compound).
• Minimal isotopic fractionation ensured by complete combustion and chromatographic integrity.

Application to Doping Control

• ERC δ13C values between –16 and –24‰ provide internal benchmarks.
• δ13C differences >3‰ between ERC and target steroids reliably indicate exogenous administration.

Benefits and Practical Applications

The integrated AnthrovisION GC-C-IRMS platform offers high sensitivity, robust precision, and streamlined workflows. Automated instrument validation and data processing ensure traceable results and compliance with WADA standards. Laboratories benefit from minimal footprint, simplified sample prep, and reliable detection of steroid misuse.

Future Trends and Possibilities

Advances in microreactor technology and IRMS detector sensitivity will lower quantification limits further, enabling detection of ultra-trace steroid metabolites. Enhanced software algorithms and machine learning could automate anomaly detection and expand isotope analysis to additional biomarkers. Integration with high-resolution mass spectrometry may offer simultaneous structural and isotopic confirmation.

Conclusion

GC-C-IRMS using the AnthrovisION solution provides a validated, high-performance method for distinguishing synthetic and endogenous steroids in urine. Its precision, sensitivity, and compliance with WADA guidelines make it an essential tool for anti-doping laboratories.

Reference

Ouellet A, LeBerre N, Ayotte C. A simplified and accurate method for the analysis of urinary metabolites of testosterone-related steroids using gas chromatography/combustion/isotope ratio mass spectrometry. Rapid Commun Mass Spectrom. 2013;27:1739–1750.