Rapid Identification and Quantification of Performance-Enhancing Stimulants in Human Urine Using High-Resolution Mass Spectrometry

Importance of the Topic

Performance-enhancing stimulants are widely abused in competitive sports to reduce fatigue and boost alertness, posing significant health risks to athletes. Rigorous detection methods are essential to enforce anti-doping regulations and safeguard athlete well-being.

Study Objectives and Overview

This study aimed to develop and validate a rapid, high-throughput workflow for the unequivocal identification and quantification of 15 structurally diverse stimulants in human urine. By combining a simple sample preparation protocol with high-resolution mass spectrometry and data-independent acquisition, the method addresses limitations of traditional HPLC-UV and GC-MS approaches.

Methodology and Instrumentation

Sample preparation involved a tenfold dilution of spiked urine with acetonitrile/methanol (80/20, v/v), followed by ultracentrifugation. Calibration curves were built over 100–1000 ng/mL using two replicates per level.

Liquid chromatography was performed on a Phenomenex C18 column (100 × 2.1 mm, 1.7 µm) at 45 °C with water and acetonitrile modifiers, a 0.5 mL/min flow rate, and a 7 min run time.

Mass spectrometry data were acquired in positive mode using SWATH® Acquisition on the SCIEX X500R QTOF System, with one TOF MS scan followed by 12 variable Q1 MS/MS windows across m/z 100–350 and a cycle time of 0.555 s. Data processing utilized SCIEX OS Software 1.5.

Instrumentation Used

• SCIEX ExionLC™ AC UHPLC system
• Phenomenex C18 column (100 × 2.1 mm, 1.7 µm)
• SCIEX X500R QTOF mass spectrometer
• SCIEX OS Software 1.5 for data acquisition and processing

Main Results and Discussion

The method achieved near-baseline chromatographic separation of all 15 stimulants within 7 min. Calibration curves exhibited excellent linearity (R2 > 0.99) and sensitivity, with limits of detection (LOD) between 1.0–30 ng/mL and limits of quantification (LOQ) between 3.3–100 ng/mL.

Matrix effects were limited (±2.2 % to ±35.7 %), indicating minimal ion suppression/enhancement. Intra-day precision and accuracy were both below 20 % across all tested levels (100, 500, 1000 ng/mL), confirming the robustness and reproducibility of the dilute-and-shoot workflow.

Benefits and Practical Applications

• High throughput: simple dilute-and-shoot protocol reduces sample preparation time.
• Comprehensive screening: SWATH acquisition archives full MS/MS spectra for each stimulant in every sample.
• Regulatory compliance: sensitive detection aligned with WADA Prohibited List requirements.
• Scalability: potential to expand the panel to additional stimulants with minimal method adjustment.

Future Trends and Applications

Advances in data-independent acquisition and HRMS hardware are expected to further enhance sensitivity and throughput. Integration with automated sample handlers and artificial intelligence–driven data analysis may enable real-time anti-doping monitoring and retrospective screening of large sample cohorts.

Conclusion

The presented workflow leverages rapid sample preparation and SWATH® acquisition on a QTOF platform to deliver reliable, high-sensitivity detection of performance-enhancing stimulants in urine. Its robust performance, combined with digital archiving of full MS/MS datasets, supports routine anti-doping applications and future panel expansions.

References

• Negri P, Gerace E, Di Corcia D, Vincenti M, Salomone A. Rapid Identification and Quantification of Performance-Enhancing Stimulants in Human Urine Using High-Resolution Mass Spectrometry. Application note, SCIEX X500R QTOF System, 2020.