Analysis of Doping Agents by UPC2-MS/MS

Importance of the Topic

Anti-doping analysis faces the challenge of detecting a broad spectrum of banned substances with diverse chemical properties. Many polar compounds exhibit poor retention and peak shape in traditional GC and LC methods. Ultra Performance Convergence Chromatography coupled with tandem mass spectrometry (UPC2-MS/MS) offers orthogonal separation, improving selectivity and enabling reliable detection of compounds that are otherwise difficult to analyze.

Objectives and Overview of the Study

This application study aimed to develop and validate a UPC2-MS/MS method for a wide panel of WADA-listed doping agents, including extremely polar analytes such as meldonium, amiloride, and ethyl glucuronide. The goals were to achieve stable retention, high sensitivity at or below WADA’s MRPL, and robust performance across authentic urine samples.

Methodology and Instrumentation

Sample Preparation:

• 200 µL urine diluted with 790 µL acetonitrile and 10 µL internal standard solution.
• Centrifugation at 5000 rcf for 10 min; 2 µL injected directly.

Chromatographic Conditions:

• System: Waters ACQUITY UPC2.
• Column: Torus Diol (130 Å, 1.7 µm, 3.0 × 100 mm) at 35 °C.
• Mobile phases: CO₂ (A) and methanol with 0.1 % strong ammonia (B); flow 1.2 mL/min; make-up flow methanol 0.2 mL/min.
• Gradient optimized for broad retention of polar and nonpolar compounds.

MS/MS Detection:

• Xevo TQ-XS triple quadrupole with ESI+ and ESI– modes.
• Compound-dependent cone and collision voltages.

Main Results and Discussion

Column and Modifier Screening identified the Torus Diol stationary phase with 0.1 % strong ammonia modifier as optimal, delivering symmetrical peaks and robust retention for all test compounds. In an expanded panel of over 30 doping agents, most analytes exhibited stable retention times with %RSD < 0.6 % over 23 batches. Polar markers such as ethyl glucuronide and meldonium were well resolved, highlighting UPC2’s complementary selectivity to LC and GC. Sensitivity experiments confirmed detection at or below WADA MRPL values; even challenging analytes like ketoconazole were consistently identified near their limits.

Benefits and Practical Applications of the Method

• Orthogonal selectivity improves coverage of polar to nonpolar compounds.
• High retention time reproducibility meets WADA identification criteria.
• Sensitivity sufficient for MRPL compliance across a diverse analyte set.

Future Trends and Potential Applications

UPC2-MS/MS can be extended to emerging performance-enhancing substances and novel metabolites. Integration with high-resolution MS and automated sample workflows may further enhance throughput and identification confidence. Inter-laboratory validation could establish UPC2 as a standardized complementary technique in anti-doping laboratories.

Conclusion

The developed UPC2-MS/MS method on the ACQUITY UPC2–Xevo TQ-XS platform provides a reliable, orthogonal approach for anti-doping analysis. It achieves stable retention, excellent sensitivity, and robust performance for a broad panel of doping agents, addressing limitations of existing GC and LC assays.

Reference

• World Anti-Doping Agency. The 2021 Prohibited List.
• WADA. TD2019MRPL Minimum Required Performance Levels, 2019.
• WADA. TD2015IDCR Criteria for Chromatographic-Mass Spec Confirmation, 2015.
• Nováková L. et al. Anal. Chim. Acta 2015, 853, 647–659.
• Losacco G. et al. Anal. Sci. Adv. 2020.