A Sensitive and Reliable Method for Anabolic Agents in Human Urine on the Agilent 7000 Triple Quadrupole GC/MS

Importance of the Topic

The detection of anabolic agents in human urine is critical for maintaining fairness and integrity in competitive sports. Highly sensitive and reliable analytical methods are essential to meet World Anti-Doping Agency (WADA) performance requirements and to identify both exogenous and endogenous metabolites at trace levels.

Study Objectives and Overview

This study aimed to develop a rapid screening protocol for seven key anabolic compounds in urine using an Agilent 7890 GC coupled to an Agilent 7000 triple quadrupole GC/MS. The method was validated against WADA Minimum Required Performance Limits (MRPLs) and applied to 1367 samples collected during the XVI Pan American Games to demonstrate robustness under real-world conditions.

Methodology and Instrumentation

Sample Preparation and Derivatization

• Urine (2.5 mL) was incubated with β-glucuronidase at 55 °C for one hour to release conjugated metabolites.
• pH was adjusted to 9.5 and analytes were extracted with tert-butyl methyl ether.
• Extracts were evaporated and derivatized with MSTFA/NH4I/ethanethiol at 60 °C for 30 minutes.

GC/MS/MS Conditions

• Instrument: Agilent 7890 GC with split/splitless inlet and 7000 Series Triple Quadrupole MS.
• Column: HP-Ultra 1 Inert, 25 m × 0.2 mm, 0.11 μm film; helium carrier at 1 mL/min.
• Oven program: 30 °C/min to 230 °C, 15 °C/min to 290 °C, 70 °C/min to 310 °C.
• Ionization: EI in Selected Reaction Monitoring mode with optimized collision energies.

Key Results and Discussion

The method achieved detection at or below MRPLs (2–10 ng/mL) for all target analytes within a 7.3-minute run time. Selectivity was confirmed by absence of interferences in six blank urine samples. Repeatability (RSD) at MRPL concentrations ranged from 4.9% to 13.4%. Over 82 analytical batches, the ratio of characteristic transitions remained constant (RSD below 7%), demonstrating exceptional ruggedness and signal-to-noise stability.

Benefits and Practical Applications

• Fast analysis throughput supports high sample volumes in doping control programs.
• Enhanced sensitivity for metabolites not detectable by conventional single-quadrupole GC/MS or LC/MS/MS.
• Compliance with WADA MRPL ensures global acceptance in accredited laboratories.

Future Trends and Opportunities

• Integration of faster GC ramps and high-throughput automation to further reduce analysis time.
• Expansion of analyte panels to include emerging synthetic steroids and designer analogs.
• Advanced data processing and machine learning to enhance detection of novel metabolites.
• Coupling with complementary techniques such as high-resolution MS for structural elucidation.

Conclusion

A robust, sensitive, and rapid GC/MS/MS method was established for anabolic agent screening in urine, meeting WADA standards and proving effective across over 1300 real samples. The Agilent 7000 Series Triple Quadrupole GC/MS System offers a powerful platform for anti-doping laboratories seeking reliable routine screening.

Instrumentation Used

• Agilent 7890 Series Gas Chromatograph with split/splitless inlet
• Agilent 7000 Series Triple Quadrupole Mass Spectrometer
• HP-Ultra 1 Inert GC Column (25 m × 0.2 mm, 0.11 μm film)
• MSTFA derivatization reagents, β-glucuronidase enzyme

References

1. Delgadillo MA et al. Sensitive and robust method for anabolic agents in human urine by gas chromatography–triple quadrupole mass spectrometry. J Chromatogr B. 2012.
2. World Anti-Doping Agency. The 2011 Prohibited List. International Standard.
3. World Anti-Doping Agency. TD2010MRPL. Minimum Required Performance Limits.
4. Baumann S. Long-term Detection of Anabolic Steroid Metabolites in Urine. Agilent Technologies Application Note.
5. Van Eenoo P et al. A fast, comprehensive screening method for doping agents in urine by gas chromatography-triple quadrupole mass spectrometry. J Chromatogr A. 2011.
6. International Conference on Harmonization. Q2B Validation of Analytical Procedures: Methodology. 1996.