Fast and Comprehensive Doping Agent Screening in Urine by Triple Quadrupole GC/MS

Importance of the topic

The detection of prohibited substances in athletes’ urine is critical for fair competition and public health
World Anti-Doping Agency (WADA) sets Minimum Required Performance Levels (MRPLs) for many drug classes
Comprehensive, rapid screening at or below MRPLs in complex matrices like urine demands advanced hyphenated methods
Combining gas chromatography with triple quadrupole mass spectrometry offers high selectivity, sensitivity and throughput

Objectives and overview of the study

Develop a single-run GC-MS/MS method to screen over 150 drugs and metabolites spanning seven prohibited classes
Aim for run times under eight minutes to accelerate sample throughput and meet WADA guidelines
Provide quantitative analysis of endogenous steroid profiles and selected urinary markers
Include qualitative detection of a broad range of anabolic agents, stimulants, narcotics, β2-agonists, hormone modulators, diuretics, beta-blockers and cannabinoids

Methodology and sample preparation

Urine hydrolyzed with β-glucuronidase (1.5 h at 56 °C) to release conjugated steroids
Liquid-liquid extraction with diethyl ether followed by evaporation of the extract
Derivatization using MSTFA-NH4I-ethanethiol (100:2:3, v/w/v), heated for 60 min at 80 °C to form volatile TMS derivatives

Used instrumentation

• Agilent 7890 gas chromatograph with split/splitless capillary inlet
• Agilent 7000A Triple Quadrupole GC/MS system
• Gerstel MPS2 autosampler and programmable temperature vaporizing (PTV) injector
• Agilent J&W HP-1 Ultra Inert capillary column (12.5 m × 0.2 mm i.d., 0.11 µm film)
• Hydrogen carrier gas at 1.0 mL/min; temperature program from 100 °C to 310 °C in three ramps
• Electron ionization; multiple reaction monitoring with optimized collision energies

Main results and discussion

The total run time was 7.98 minutes, enabling high-throughput analysis
Quantitative determination of key endogenous steroids (T, E, A, Et, DHT, DHEA, androstenedione, 5αab, 5βab) with R2 ≥ 0.96
Qualitative MRM detection of 41 anabolic metabolites, 4 other anabolics, 6 β2-agonists, 11 hormone modulators, 19 narcotics, 16 stimulants, 15 beta-blockers and THC-COOH
Limits of detection at or below WADA MRPLs for nearly all target analytes; alternative compliant metabolites included when needed
Baseline separation of critical isomer pairs (androsterone vs etiocholanolone) supports reliable quantification
Internal standards covering free and glucuronidated steroids monitor hydrolysis, extraction and derivatization efficiency per sample

Benefits and practical application of the method

Reduces sample volume to 1 mL urine versus 2–5 mL in conventional assays
Combines quantitative steroid profiling and broad qualitative screening in one rapid analysis
Meets WADA requirements for surveillance and forensic toxicology reporting within 24–48 h turnaround
Enhances laboratory efficiency by minimizing multiple sample preparations and instrument methods

Future trends and opportunities

Integration of high-resolution mass spectrometry for non-target and retrospective analysis
Extension to alternative matrices such as blood, saliva and hair for longitudinal monitoring
Automation of sample preparation and on-line derivatization to further increase throughput
Use of sustainable carrier gases and miniaturized chromatography to reduce environmental impact
Application of data‐driven software and machine learning for streamlined data interpretation and anomaly detection

Conclusion

A robust GC-MS/MS methodology on Agilent 7000A allows fast, comprehensive screening of over 150 doping agents
Achieves quantitative steroid profiling and sensitive detection at or below WADA MRPLs in under eight minutes
Offers a practical platform for anti-doping and forensic laboratories seeking high throughput, reliable performance

References

1. van Eenoo P, Van Gansbeke W, De Brabanter N, Deventer K, Delbeke FT. A fast, comprehensive screening method for doping agents in urine by gas chromatography-triple quadrupole mass spectrometry. J Chromatogr A. 2010 Oct 8.
2. World Anti-Doping Agency. Technical Document on Minimum Required Performance Levels (MRPLs), 2010.
3. Parr MK, Fußhöller G, Schlörer N, Opfermann G, Piper T. Metabolism of androsta-1,4,6-triene-3,17-dione and detection by GC/MS in doping control. Rapid Commun Mass Spectrom. 2009;23:207–218.
4. EURACHEM. The Fitness for Purpose of Analytical Methods; A Laboratory Guide to Method Validation. 1998.
5. Henze MK, Opfermann G, Spahn-Langguth H, Schänzer W. Screening of β2-agonists and confirmation of fenoterol, orciprenaline, reproterol and terbutaline with GC/MS as tetrahydroisoquinoline derivatives. J Chromatogr B. 2001;751:93–105.
6. Kiousi P, Angelis YS, Lyris E, Koupparis M, Calokerinos AC, Atta-Politou J, Georgakopoulos CG. Two-step silylation procedure for unified analysis of 190 doping control substances in human urine samples by GC–MS. Bioanalysis. 2009;1:1209–1224.
7. World Anti-Doping Agency. TD2010MRPL. 2010.