Quantitation of Ethyl Glucuronide and Ethyl Sulfate in Urine using LC-MS/MS

Importance of the Topic

Monitoring alcohol consumption through biochemical markers is essential in clinical toxicology, rehabilitation programs, and legal settings. Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are reliable indicators of recent alcohol intake, offering longer detection windows than ethanol itself. A robust and sensitive analytical method for quantifying these metabolites in urine supports zero‐tolerance policies and improves patient monitoring.

Study Objectives and Overview

This work presents a streamlined LC-MS/MS assay using a Shimadzu Nexera LC coupled to the LCMS-8045 triple-quadrupole mass spectrometer. The primary goals were to develop and validate a rapid, accurate, and reproducible method for simultaneous quantitation of EtG and EtS in human and synthetic urine matrices.

Methodology and Instrumentation

Sample preparation involved spiking pooled human urine and synthetic urine (“surine”) with EtG, EtS, and deuterated internal standards (EtG-d5, EtS-d5) at seven calibration levels (50–5000 ng/mL) plus four QC levels (50, 125, 700, 4000 ng/mL). Following vortexing and centrifugation, 10 µL of supernatant was injected.

Used Instrumentation

• Shimadzu Nexera UHPLC system with Raptor EtG/EtS column (100 × 2.1 mm, 2.7 µm) and 0.2 µm precolumn filter
• Shimadzu LCMS-8045 triple-quadrupole mass spectrometer
• Negative-mode electrospray ionization with multiple reaction monitoring (MRM)

Main Results and Discussion

Baseline separation of EtG (0.81 min) and EtS (1.87 min) was achieved, free from significant matrix interferences observed at 0.5 min and 1.2 min in urine. Calibration curves displayed excellent linearity (R² > 0.998) in both matrices. Accuracy for QC levels ranged from 88 % to 114 % in human urine and 88 % to 102 % in surine. Analysis of certified reference (UTAK) controls and positive in-house samples demonstrated consistent quantitation with minimal matrix effect.

Benefits and Practical Applications

• High throughput: 4-minute run time per sample
• Sensitive detection: LLOQ of 50 ng/mL
• Reliable performance across different urine matrices
• Applicability in clinical, forensic, and rehabilitation testing

Future Trends and Opportunities

Advancements may include miniaturized microflow LC-MS platforms for even faster screening, incorporation of high-resolution mass spectrometry to confirm novel ethanol metabolites, and integration with automated sample preparation to increase laboratory throughput. Expansion to blood and hair matrices can broaden diagnostic coverage.

Conclusion

The described LC-MS/MS method using the Shimadzu LCMS-8045 reliably quantifies EtG and EtS in urine and surrogate matrices with high accuracy, precision, and speed. Its robustness and simplicity make it well suited for routine laboratory workflows in toxicology and compliance monitoring.