Analysis of Doping and Forensic Drugs in Urine Using High-Resolution GC/Q-TOF

Significance of the topic

In anti-doping control and forensic toxicology, analysts face increasing pressure to detect and quantify trace levels of prohibited substances in complex biological matrices. Traditional GC triple quadrupole instruments offer sensitivity and dynamic range but lack comprehensive screening and retrospective analysis capabilities. High-resolution accurate-mass GC/Q-TOF systems combine sensitive targeted quantitation with post-run data mining, enabling both strict regulatory compliance and the discovery of unexpected compounds.

Objectives and Study Overview

This study evaluates the performance of an Agilent 7250 GC/Q-TOF with a low-energy electron ionization source for simultaneous quantitative analysis at World Anti-Doping Agency Minimal Required Performance Levels (MRPL) and untargeted screening of doping and forensic drugs in urine. Two phases were conducted: sensitivity and mass accuracy assessment in spiked urine at MRPL levels, followed by quantitative and untargeted analysis of six diverse urine samples spiked with 28 representative compounds.

Methodology

Urine samples were extracted, dried and derivatized with a MSTFA/NH4I/ethanethiol mixture. An accurate-mass Personal Compound Database and Library (PCDL) guided automated quantitation method building. Data were processed using MassHunter Quantitative Analysis (version B.09) and Qualitative Analysis (version B.08) with Unknowns Analysis for feature detection and NIST14.L library searching.

Instrumentation

The analytical platform consisted of an Agilent 7890B GC coupled to a 7250 GC/Q-TOF equipped with a low-energy EI source. Key GC parameters included an HP-1MS 12 m × 0.25 mm × 0.25 µm column, splitless injection of 1 µL, and a multi-step oven program from 110 °C to 320 °C. The TOF was operated at 50–650 m/z, 3–5 Hz acquisition rate, with EI at 70 eV and reduced energies (17 eV, 15 eV).

Main Results and Discussion

Mass accuracy for characteristic ions in solvent and spiked urine at MRPL levels was generally below 1 ppm. Lowering electron energy enhanced molecular ion signals for analytes with weak molecular peaks at 70 eV. Quantitative evaluation across six donors showed relative standard deviations under 20% and calibration linearity (R2>0.99) for 22 compounds at or above MRPL. An independent calibration for epitestosterone demonstrated linearity from 1 to 500 ng/mL (R2=0.999). Untargeted Unknowns Analysis detected more than 20 additional drugs and metabolites—including stimulants, diuretics, corticosteroids, and sedatives—via library matching and accurate-mass features.

Benefits and Practical Applications

• Integrated targeted quantitation and retrospective screening in a single run.
• High sensitivity and wide dynamic range at or below regulatory MRPL values.
• Enhanced molecular ion detection using low-energy EI for confident compound identification.
• Streamlined workflow with automated PCDL-based method generation and untargeted feature discovery.

Future Trends and Applications

The demonstrated GC/Q-TOF approach paves the way for expanded retrospective data mining in anti-doping and forensic labs. Future developments may include larger accurate-mass libraries, advanced algorithms for feature prioritization, and integration with high-throughput sample preparation platforms. Low-energy ionization strategies will further improve molecular structure elucidation and support broader applications in clinical toxicology and environmental monitoring.

Conclusion

This work confirms that high-resolution GC/Q-TOF with low-energy EI meets strict sensitivity, accuracy, and dynamic range requirements for doping control and forensic screening. The combined quantitative and untargeted strategy enhances detection capabilities, offers retrospective analysis, and supports regulatory compliance and investigative toxicology with a single analytical platform.

References

1. Van Gansbeke W, Polet M, Hooghe F, Devos C, Van Eenoo P. Improved sensitivity by use of gas chromatography-positive chemical ionization triple quadrupole mass spectrometry for the analysis of drug related substances. Journal of Chromatography B. 2015;1001:221–240.
2. WADA. TDMRPL2018: minimum required performance levels for detection and identification of non-threshold substances. 2018.