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

Importance of the Topic

Polar and ionic drugs and metabolites such as GHB, GBL, ethyl sulfate (ETS), ethyl-β-D-glucuronide (ETG), GHB-β-O-glucuronide, meldonium and γ-butyrobetaine present significant analytical challenges in doping control due to poor retention and peak shape in conventional HPLC or GC methods. Supercritical fluid chromatography (SFC) with ion-exchange stationary phases offers complementary selectivity and robust quantification of these analytes in biological matrices.

Objectives and Study Overview

This study aimed to develop and validate an SFC/MS/MS method using the Agilent 1260 Infinity II SFC System and triple quadrupole mass spectrometry to separate and detect highly polar and ionic compounds relevant to anti-doping analysis. Method development included stationary phase screening, modifier and additive optimization, and evaluation of chromatographic parameters under SFC conditions.

Methodology and Instrumentation

• Sample preparation: protein precipitation of urine with methanol, 1:10 final dilution.
• Instrumentation:
  • Agilent 1260 Infinity II SFC System (Control Module, Binary Pump, Multisampler, Diode Array Detector, Multicolumn Thermostat)
  • Agilent 6470A Triple Quadrupole LC/MS with Agilent Jet Stream
  • Data processing: Agilent MassHunter software with MRM and source optimizers
• Stationary phase: silica-based strong cation exchange (SCX) with phenyl modification (4.6×150 mm, 5 µm)
• Mobile phase: CO2 with methanol/water (95:5 v/v) containing 20 mM ammonium formate and 15 mM formic acid
• Gradient: 15% modifier to 60% in 1 min; flow 2.0 to 2.5 mL/min; column 45 °C; backpressure 170 bar
• MS conditions: ESI+ and ESI−, optimized MRM transitions, drying gas 250 °C, sheath gas 375 °C, nebulizer 15 psi

Main Results and Discussion

The optimized SFC method achieved baseline separation of all seven analytes within 4 minutes. Retention times and peak shapes were controlled by water content and additive concentration; higher ammonium formate improved peak shape, particularly for ETG. Method performance data: detection limits ranged from 0.001 mg/L (ETS, meldonium) to 0.5 mg/L (ETG, GHB-Gluc); LOQs were comparable or superior to conventional HPLC/MS/MS methods. Linearity (R2 > 0.97), intra-day precision (area CV 2.1–13.4%, RT CV < 1%), and matrix factors (0.30–1.57) demonstrated robust quantification in urine. LOQs met or exceeded doping control cut-off levels and MRPL requirements.

Benefits and Practical Applications

• Orthogonal separation to HPLC, retaining polar and ionic analytes without breakthrough
• Rapid analysis (< 5 min) with high resolution and consistent peak shapes
• Robust quantification at sub-µg/L levels for anti-doping screening
• Green solvent usage through CO2 as main mobile phase component

Future Trends and Potential Applications

• Extension to broader panels of polar biomarkers and metabolites
• Integration of high-resolution mass spectrometry for non-targeted screening
• Automation of sample preparation and on-line SPE coupling
• Application in clinical and forensic toxicology for rapid polar compound profiling

Conclusion

A dedicated SFC/MS/MS workflow using a strong cation exchange column and optimized methanol/water modifier has been established for efficient separation and quantification of highly polar and ionic doping control analytes. The method delivers rapid, precise, and sensitive analysis in urine, offering a viable alternative to traditional HPLC and GC techniques for routine anti-doping and forensic applications.

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