Analysis of Polar and Ionic Drugs in Doping Control by Ion‑Exchange Chromatography with the Agilent 1260 Infinity II SFC System

Importance of Topic

The analysis of highly polar and ionic drugs and metabolites is a persistent challenge in forensic and doping-control laboratories. Traditional techniques such as UHPLC/MS/MS and GC/MS/MS often struggle with retention and peak shape for these analytes. Supercritical fluid chromatography (SFC) coupled with ion-exchange stationary phases offers an orthogonal approach that can improve separation and quantification reliability under lower solvent consumption and faster run times.

Objectives and Study Overview

This study aimed to develop and optimize an SFC/MS/MS method using the Agilent 1260 Infinity II SFC System for seven target compounds relevant in doping control: GBL, GHB, ETS, ETG, GHB-Gluc, Meldonium, and G-BTB. Key goals included evaluating stationary phases, modifier/additive combinations, gradient conditions, and system parameters to achieve baseline separation, acceptable sensitivity, and quantitative performance equivalent or superior to conventional HPLC/MS/MS assays.

Instrumentation

• Agilent 1260 Infinity II SFC Control Module
• Agilent 1260 Infinity II SFC Binary Pump
• Agilent 1260 Infinity II SFC Multisampler
• Agilent 1260 Infinity II Diode Array Detector with high-pressure SFC flow cell
• Agilent 1260 Infinity II Multicolumn Thermostat
• Agilent 6470A Triple Quadrupole LC/MS with Jet Stream technology

Methodology

Sample preparation involved protein precipitation of urine with methanol and a 1:10 final dilution. A silica-based strong cation-exchange column (4.6×150 mm, 5 µm) modified with sulfonic acid and phenyl groups was selected. The mobile phase consisted of CO₂ with 15% MeOH/H₂O (95/5) buffered with 20 mM ammonium formate and 15 mM formic acid. Gradient elution ramped from 15% to 60% modifier in 1 minute, coupled with a flow change from 2.0 to 2.5 mL/min. Column temperature was maintained at 45 °C and backpressure at 170 bar. Electrospray ionization in both polarities was used, with optimized MRM transitions and source parameters via Agilent MassHunter tools.

Main Results and Discussion

Screening of 12 stationary phases and nine modifier combinations identified the SCX phase with methanol/water buffer as optimal. Increasing additive concentration improved peak shape, particularly for ETG. Gradient and flow adjustments reduced retention times and sharpened peaks. The final method achieved baseline separation of all analytes in under 6 minutes. LODs ranged from 0.001 to 0.5 mg/L and LOQs from 0.005 to 2.5 mg/L, meeting or exceeding existing HPLC/MS/MS performance. Retention time precision was <1% CV, and area precision <14% CV. Matrix effects were minimal to moderate and compensated via calibration.

Benefits and Practical Applications

• Reliable quantification of polar/ionic analytes in doping control and forensic matrices.
• Comparable sensitivity to HPLC/MS/MS with shorter run times and reduced solvent use.
• Orthogonal selectivity to conventional reversed-phase methods.

Future Trends and Potential Applications

The integration of ion-exchange SFC with high-resolution MS could expand to additional polar biomarkers and environmental contaminants. Continued development of tailored stationary phases and greener mobile phases will further enhance throughput, sensitivity, and robustness in clinical, forensic, and food-safety analyses.

Conclusion

This work demonstrates a fast, sensitive, and robust SFC/MS/MS workflow for the analysis of challenging polar and ionic compounds in urine. The optimized method offers clear separation, low detection limits, and performance on par with or superior to conventional HPLC approaches, supporting its adoption in doping-control and forensic laboratories.

References

1. Par MK et al. Anal. Bioanal. Chem. 2016;408:6789–6797.
2. Desfontaine V et al. Bioanalysis 2015;7:1193–1195.
3. Fujito Y et al. J. Chromatogr. A 2017;1508:138–147.
4. Novakova L et al. Anal. Chim. Acta 2015;853:647–659.
5. Abanades S et al. Ann. NY Acad. Sci. 2006;1074:559–576.
6. Mehling LM et al. Forensic Toxicol. 2017;35:263–274.
7. Xhaferaj M et al. J. Chromatogr. A 2020;1614:460726.
8. Albertmann M et al. J. Chromatogr. Sci. 2012;50:51–56.
9. Kim Y et al. Mass Spectrom. Lett. 2017;8:39–43.
10. WADA Technical Document TD2016EAAS 2015.
11. Brailsford AD et al. J. Anal. Toxicol. 2012;36:88–95.
12. WADA TD2018MRPL 2018.