Quantitative LC/MS/MS Analysis of Ethyl Glucuronide and Ethyl Sulfate using Charged Surface-C18 Column

Importance of the Topic

Ethyl Glucuronide (EtG) and Ethyl Sulfate (EtS) are key biomarkers for detecting recent alcohol consumption. Their polar nature makes retention on traditional reversed-phase columns challenging. Reliable, high-throughput quantification supports forensic investigations, clinical monitoring and workplace safety programs.

Objectives and Study Overview

This study evaluated a hybrid end-capped charged surface C18 column for LC/MS/MS quantification of EtG and EtS in urine. The goals were to optimize sample dilution and injection volume, assess chromatographic performance, validate calibration linearity and evaluate method robustness over extensive injection series.

Methods and Instrumentation

Sample Preparation

• Urine spiked with EtG and EtS standards and corresponding deuterated internal standards
• Dilution of 1 to 50 in mobile phase A (0.01 formic acid in water)
• Direct injection (“dilute and shoot”) without further cleanup

Chromatography and Detection

• Column temperature 40 °C; mobile phase A water with 0.01 formic acid, B methanol; flow rate 0.35 mL/min
• Gradient from 2 B to 98 B in 4 minutes on Agilent Poroshell CS-C18 (2.1×50 mm or 2.1×100 mm, 2.7 µm)
• Triple quadrupole MS in negative ion mode with MRM transitions for EtG, EtS and their d5 internal standards
• 7-point calibration curves (EtG 100–10 000 ng/mL, EtS 10–1 000 ng/mL) using 1/x weighting

Instrumentation Employed

• Agilent 1290 Infinity II Binary Pump G7120A
• Agilent 1290 Infinity II Multisampler G7167B
• Agilent 1290 Infinity II Multicolumn Thermostat Column Compartment G7116B
• Agilent 6470 LC/TQ Mass Spectrometer G6470B
• Agilent Ultivo LC/TQ Mass Spectrometer G6465B
• Agilent Poroshell InfinityLab CS-C18 Columns (2.1×50 mm and 2.1×100 mm, 2.7 µm)

Main Results and Discussion

The charged surface C18 column achieved clear retention and separation of both analytes despite their hydrophilicity. A 1 to 50 dilution combined with a 2 µL injection volume delivered optimal peak shape and signal response. Calibration curves were linear with R2 > 0.998 for both EtG and EtS. Retention time and peak area remained stable over 800+ injections. Reproducibility was confirmed by 1 000 injections across five urine samples, including negatives and varying analyte levels, demonstrating consistent quantification.

Benefits and Practical Applications

The method’s rapid 4 minute run time and minimal sample preparation enable high-throughput workflows. Robust performance and straightforward operation make it suitable for routine forensic, clinical and workplace alcohol monitoring, as well as QA/QC laboratories.

Future Trends and Application Opportunities

Further studies on column lifetime and long-term robustness will support method validation for regulated environments. Integration with automated sample handling and extension to other polar metabolites are promising avenues. Adoption of charged surface chemistries may expand to broader panels of endogenous and exogenous small molecules.

Conclusion

The charged surface C18 LC/MS/MS method provides reliable, reproducible quantification of EtG and EtS with minimal preparation and fast throughput. It offers a practical solution for high-volume testing and lays the groundwork for future automation and method expansion.