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

Significance of the topic

The detection of highly polar performance enhancing substances in biological matrices is critical for fair sport and public health. Emerging designer agents and metabolic conjugates pose analytical challenges due to polarity and structural similarity. A robust screening method for compounds such as meldonium and various adrenergic agents improves anti doping efforts worldwide.

Objectives and Study Overview

This study aimed to develop a two dimensional hydrophilic interaction liquid chromatography tandem mass spectrometry workflow for simultaneous detection of six polar doping agents and their sulfate metabolites in urine. The method focuses on performance evaluation over a wide concentration range and on isomer differentiation such as norfenefrine and octopamine.

Methodology and Instrumentation

The sample preparation involved protein precipitation of urine followed by direct injection onto a two dimension HILIC system. The first dimension used a trapping column for cleanup and preconcentration, followed by analytical separation on a Nucleodur HILIC column. Rapid polarity switching in the mass spectrometer enabled detection of both positive and negative ion sulfated metabolites. Instrumentation used included a Nexera X2 LC system and LCMS 8060 mass spectrometer with electrospray ionization.

• Analytical column Nucleodur HILIC 100 mm by 2 mm id 1.8 mu m
• Trapping column Nucleodur HILIC 20 mm by 2 mm id 3 mu m
• Mobile phases water and acetonitrile with ammonium acetate buffer
• Flow programming for two dimension switching and gradient elution
• Mass spectrometry MRM transitions for parent compounds and sulfated forms

Main Results and Discussion

Calibration of six target analytes from 1 to 200 ng per mL yielded linear responses with correlation coefficients above 0.997. The method successfully quantified meldonium, synephrine, norfenefrine, etilefrine, oxilofrine and octopamine along with their conjugates. Detection of sulfated metabolites in both positive and negative mode allowed confirmation of parent peaks. Isomeric compounds norfenefrine and octopamine were resolved based on their specific sulfate signals under negative ion conditions.

Benefits and Practical Applications

• High sensitivity and selectivity for a range of polar doping agents
• Comprehensive screening that includes both free and conjugated forms
• Rapid polarity switching avoids additional sample processing
• Compatibility with anti doping laboratory workflows for routine analysis

Future Trends and Potential Use

Continued method development may extend this workflow to novel designer agents and additional metabolites. Advances in column chemistry and high resolution mass spectrometry could further improve specificity. Integration with automated sample handling will enhance throughput in anti doping laboratories.

Conclusion

The two dimensional HILIC MSMS approach provides a powerful tool for detecting highly polar doping substances and their metabolites in urine. Its high linearity, sensitivity and ability to distinguish isomers support its application in sports drug testing and regulatory screening.

References

• Anal Bioanal Chem 2015 407 5354-5379
• Drug Test Analysis 2015 7 973-979