Measuring alcohol biomarkers from urine – a splitting headache!

Importance of the Topic

Monitoring ethanol metabolites in urine such as ethyl sulfate (EtS) and ethyl glucuronide (EtG) provides a non-invasive, sensitive approach to detect recent alcohol consumption in clinical, forensic and quality-control settings. Unlike blood or breath tests, these minor metabolites have longer detection windows of several days, making them valuable markers to confirm abstinence or study exposure in research programs.

Study Goals and Overview

• Address a severe matrix-induced peak splitting issue for EtS and its labeled standard observed on a competitor biphenyl UHPLC column.
• Evaluate the performance of the Thermo Scientific Hypersil GOLD VANQUISH aQ UHPLC column as an alternative stationary phase for EtS and EtG analysis.
• Characterize linearity, accuracy and precision of the revised assay under new chromatographic conditions.

Methodology and Instrumentation

Sample Preparation:

• Dilute 100 µL urine with 900 µL 0.5 % formic acid in water.
• Perform solid-phase extraction using Thermo Scientific SOLA SCX 96-well plate in non-retentive mode to remove basic and lipophilic interferences.
• Collect the eluent directly for injection.

Chromatography and Mass Spectrometry:

• UHPLC system: Thermo Scientific Vanquish Horizon with active pre-heater and still-air mode.
• Column: Hypersil GOLD VANQUISH aQ, 100 × 2.1 mm, 1.9 µm; isocratic mobile phase of 0.5 % formic acid in water; flow 0.2 mL/min; run time 7.5 min; column temperature 20 °C; injection volume 10 µL.
• MS/MS: Thermo Scientific TSQ Quantiva triple quadrupole; H-ESI negative mode; spray voltage 3000 V; vaporizer 400 °C; sheath gas 50 Arb; auxiliary gas 15 Arb; ion transfer tube 350 °C; CID gas 1.5 mTorr.
• Analyte transitions: EtG 221 → 75 m/z (CE 15.1 V); EtS 125 → 97 m/z (CE 17 V); deuterated standards accordingly.

Main Results and Discussion

A competitor biphenyl column exhibited pronounced peak splitting of EtS and its d5 standard due to matrix effects in multiple urine sources. Switching to the Hypersil GOLD VANQUISH aQ phase eliminated splitting and delivered sharp, symmetric peaks free of interferences.
Calibration and Performance:

• EtS linear range: 20–2000 ng/mL, R² = 0.9963.
• EtG linear range: 100–10000 ng/mL, R² = 0.9992.
• Accuracy bias: EtS within –7.4 % to +3.8 %; EtG within –2.1 % to +3.6 %.
• Precision (CV): ≤5.5 % for EtS; ≤5.8 % for EtG.

Benefits and Practical Applications

• Rapid, pass-through SPE simplifies cleanup and removes urobilin and other matrix components.
• Isocratic, 100 % aqueous elution reduces solvent complexity and runtime.
• Robust separation on Hypersil GOLD VANQUISH aQ ensures reliable quantitation in diverse urine matrices.
• Suitability for high-throughput clinical, forensic or industrial laboratories requiring sensitive alcohol biomarker analysis.

Future Trends and Potential Uses

• Extension to other polar or conjugated metabolites in biological fluids.
• Integration with automated SPE-UHPLC workflows for increased throughput.
• Exploration of mixed-mode stationary phases to mitigate diverse matrix effects.
• Application in population studies or occupational monitoring for comprehensive alcohol exposure profiling.

Conclusion

Replacing a biphenyl column with the Hypersil GOLD VANQUISH aQ phase successfully resolved a critical peak splitting issue for EtS and EtG in urine. The simplified SPE protocol, robust UHPLC-MS/MS conditions and excellent analytical performance support routine implementation of this method for reliable alcohol biomarker quantitation.

References

• Woodmansey K, Taylor C. Technical Note 21989. Thermo Fisher Scientific; 2020.