Screening Analysis of Steroid Profiles and Qualitative Doping Substances by GC-MS/MS

Importance of the Topic

Anti-doping screening is vital for ensuring athlete health and fair competition. The growing volume of urine samples and the introduction of the Athlete Biological Passport (ABP) underscore the need for high-throughput, highly sensitive methods. Combining qualitative detection of low-level prohibited substances with quantitative steroid profile monitoring in a single analysis streamlines workflows in WADA-accredited laboratories.

Goals and Overview

This study evaluated a single-injection GC-MS/MS method using the Shimadzu GCMS-TQ8050 NX to simultaneously quantify 180 substances, including endogenous steroids for ABP monitoring and low-level doping agents. The method aimed to detect compounds at or below WADA MRPL values while avoiding saturation for abundant endogenous steroids during international sports games in 2021.

Methodology and Instrumentation

Sample Preparation

• 2 mL urine fortified with internal standards
• SPE cleanup on Bond-Elut NEXUS
• β-Glucuronidase hydrolysis, liquid-liquid extraction into diethyl ether
• Dry-down and TMS derivatization (MSTFA/iTMS/DTE)

GC-MS/MS Conditions

• Injection: split 11 : 1, 280 °C, 2 µL
• Column: HP-ULTRA 1 (17 m × 0.20 mm × 0.11 µm)
• Carrier: helium, constant pressure (~156 kPa)
• Oven: 180 °C (1 min) → 3 °C/min → 229 °C → 40 °C/min → 300 °C (3 min), total 22 min
• Ion source 230 °C, interface 300 °C, detector voltage 0.4 kV
• Acquisition: dynamic multiple reaction monitoring (MRM)

Main Results and Discussion

Steroid Profiles

• Six endogenous steroids (androsterone, etiocholanolone, testosterone, epitestosterone, 5α/5β-androstane-3α,17β-diol) quantified over wide linear ranges with R² > 0.998.
• Carryover tests for androsterone and etiocholanolone at 10 000 ng/mL showed no residual peaks in subsequent blanks.
• Control charts for T/E ratios remained within ±2 SD across 30 batches, demonstrating robustness.

Qualitative Screening

• Twelve representative low-level analytes (e.g., 3′-hydroxy-stanozolol, clenbuterol, DHCMT m2) detected down to one-quarter of 2019 MRPL values.
• Carboxy-THC monitored before and after heavy use periods, confirming stable detection at 150 ng/mL.

Robustness and Throughput

• Twelve GCMS-TQ8050 NX units processed ~6800 samples over five months with zero detector downtime.
• Patented ion source shields minimized contamination, enabling only GC-side maintenance.
• Detector gain remained stable throughout extended high-volume operation.

Benefits and Practical Applications

The single-method approach simplifies workflows by combining qualitative and quantitative analyses in a 22-minute run. High sensitivity meets WADA MRPL requirements, and reduced maintenance ensures continuous operation during major events. The method supports both initial testing procedures and ABP monitoring in anti-doping laboratories.

Future Trends and Opportunities

Advances may include expanding target analyte lists, integrating real-time data evaluation, further automation of sample preparation, and coupling with high-resolution MS for improved selectivity. Portable GC-MS platforms could extend on-site testing capabilities.

Conclusion

The validated GCMS-TQ8050 NX workflow delivers robust, sensitive, and high-throughput screening of 180 substances, addressing both steroid profiling and low-level doping detection. Its performance during the 2021 international sports games highlights its suitability for WADA-accredited laboratories.

Used Instrumentation

• GCMS-TQ8050 NX
• AOC-20i Plus Auto Injector
• AOC-20s Plus Auto Sampler
• HP-ULTRA 1 capillary column

References

1) World Anti-Doping Agency, Annual Report, 2021
2) WADA Technical Document TD2019MRPL, 2019
3) WADA Technical Document TD2022MRPL, 2021
4) WADA, Athlete Biological Passport and Steroid Profiles Review, 2021