Quantitation of Urinary Ethyl Glucuronide and Ethyl Sulfate Using Ultrahigh Resolution LC-MS

Significance of the Topic

Ethyl glucuronide (EtG) and ethyl sulfate (EtS) serve as reliable urinary markers for recent alcohol consumption. Their extended detection window and high specificity make them indispensable in forensic toxicology for applications such as impaired driving investigations, workplace testing, and clinical monitoring.

Objectives and Study Overview

This study demonstrates a fast, accurate liquid chromatography–mass spectrometry (LC-MS) approach using ultrahigh resolution for simultaneous quantitation of EtG and EtS in urine. The goal is to validate method performance on a Thermo Scientific Exactive bench-top instrument and assess its suitability for routine forensic analysis.

Methodology and Instrumentation

Sample Preparation:

• Calibration standards prepared by spiking blank urine to 25–20 000 ng/mL.
• Internal standards EtG-d5 and EtS-d5 added to all samples.
• Samples diluted tenfold with LC mobile phase before injection.

Chromatography:

• System: Thermo Scientific Accela HPLC.
• Column: Hypersil GOLD C18, 50 × 2.1 mm, 5 µm.
• Injection volume: 20 µL diluted sample.
• Gradient run time: 6 minutes.

Mass Spectrometry:

• Instrument: Thermo Scientific Exactive bench-top LC-MS.
• Ionization: Electrospray (ESI).
• Acquisition: Full-scan at 100 000 FWHM resolution (m/z 200).
• Mass tolerance for quantitation: ±5 ppm.

Results and Discussion

The method yielded linear response for both analytes over 100–20 000 ng/mL. Key performance metrics were:

• Limit of detection: 25 ng/mL.
• Limit of quantitation: 100 ng/mL.
• Recovery: >85%.
• Precision (RSD): <15%.
• Carryover at LLOQ: <1%.
• Sample volume required: 100 µL.
• Analysis time per run: 6 minutes.

Chromatograms at the LOD showed clear peaks for EtG (RT ~1.94 min) and EtS (RT ~2.59 min) alongside their deuterated analogs, confirming specificity and signal integrity.

Benefits and Practical Applications

This ultrahigh-resolution LC-MS method offers rapid turnaround, high throughput, and excellent sensitivity for forensic urine analysis. It supports accurate casework decisions, compliance monitoring, and clinical research by minimizing sample preparation and maximizing data quality.

Future Trends and Opportunities

Advances may include integration of high-resolution tandem MS for improved structural confirmation, automated sample handling for increased throughput, untargeted screening workflows to detect novel biomarkers, and development of portable high-resolution instruments for field applications. Enhanced data processing algorithms will further streamline quantitation and reporting.

Conclusion

The validated six-minute LC-MS method on the Exactive bench-top platform delivers robust, sensitive, and precise quantitation of urinary EtG and EtS, meeting the stringent demands of modern forensic toxicology laboratories.