Screening Analysis of Highly Polar Doping Agents in Urine Using 2DLC/MS/MS

Importance of the topic

Detecting performance-enhancing and designer doping agents in urine is critical for fair play and athlete safety. Highly polar compounds such as meldonium and adrenergic stimulants often escape conventional analyses. Implementing a robust two-dimensional liquid chromatography coupled with tandem mass spectrometry (2D LC/MS/MS) approach addresses these challenges by enhancing retention, separation and sensitivity for polar analytes.

Objectives and study overview

This application focuses on simultaneously screening six polar doping agents and their relevant metabolites in human urine. The targets include the anti-ischemic drug meldonium and adrenergic agents synephrine, norfenefrine, etilefrine, oxilofrine and octopamine. The study evaluates method performance for quantitation, confirmation of sulphate conjugates and differentiation of positional isomers.

Methodology and instrumentation

The protocol employs a 2D-HILIC setup:

• Trapping column: Nucleodur HILIC (20 mm × 2 mm I.D., 3 µm) for on-line sample cleanup and pre-concentration.
• Analytical column: Nucleodur HILIC (100 mm × 2 mm I.D., 1.8 µm) for high-resolution separation.
• Mobile phases: aqueous and acetonitrile solvents modified with ammonium acetate buffer (200 mM) and acetic acid.
• Gradient and flow control: two pumps for trapping (A/B) and one pump for elution (C), with rapid polarity switching between positive and negative electrospray ionization (ESI).
• Mass spectrometer: Shimadzu LCMS-8060 with ESI source, optimized gas flows, interface temperature at 400 °C, and multiple reaction monitoring (MRM).

Sample preparation involves protein precipitation of urine with acetonitrile containing an internal standard (meldonium-d3), centrifugation and direct injection of the supernatant.

Key results and discussion

Calibration curves for all six parent compounds showed excellent linearity over 1–200 ng/mL (r² > 0.997). Limits of detection supported WADA’s reporting criteria for meldonium (> 100 ng/mL). MRM transitions enabled selective detection of parent drugs and sulphate metabolites. Rapid polarity switching confirmed the presence of conjugates and resolved isobaric compounds. In spiked and post-administration urine, synephrine, etilefrine and oxilofrine were quantified accurately. Norfenefrine and octopamine, which co-elute and share MRM transitions, were distinguished unambiguously by detecting their negative-ion sulphate metabolites.

Benefits and practical applications

The 2D HILIC approach offers:

• Improved retention and peak shape for polar analytes.
• Enhanced selectivity through orthogonal trapping and analytical separation.
• High throughput with direct injection and automated polarity switching.
• Reliable confirmation of parent drugs and metabolites in a single run.

This method is directly applicable to anti-doping laboratories for routine screening and confirmation under WADA guidelines.

Future trends and potential applications

Emerging directions include integrating high-resolution accurate-mass spectrometry for non-targeted screening, automating data analysis with machine learning algorithms, and expanding the method to cover novel designer substances and peptide hormones. Miniaturized microfluidic LC platforms may further reduce sample volume and analysis time.

Conclusion

The described 2D LC/MS/MS workflow provides a sensitive, selective and versatile solution for screening highly polar doping agents and their metabolites in urine. Its ability to confirm sulphate conjugates and resolve isomers makes it a valuable tool for modern anti-doping testing.

Reference

1. Analytical and Bioanalytical Chemistry, 2015, 407, 5354–5379.
2. Drug Testing and Analysis, 2015, 7, 973–979.