Quantification of ethyl glucuronide and ethyl sulfate in human urine by LC-HRAM(MS) for clinical research

Significance of the Topic

Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are direct biomarkers of ethanol consumption, detectable in urine long after alcohol intake. Sensitive and selective quantification of these metabolites supports clinical research, forensic investigations, and workplace testing by providing reliable evidence of recent alcohol use.

Objectives and Study Overview

This study aimed to develop and validate a rapid, robust analytical workflow for the quantification of EtG and EtS in human urine using high-resolution accurate-mass liquid chromatography–mass spectrometry (LC-HRAM MS). The method integrates a full-scan (FullMS) and parallel reaction monitoring (PRM) acquisition strategy on a Q Exactive Plus Orbitrap instrument to achieve high specificity and sensitivity.

Methodology and Used Instrumentation

Sample Preparation

• ClinMass LC-MS/MS Complete Kit for Ethylglucuronide and Ethylsulfate in Urine (RECIPE Chemicals + Instruments)
• Fifty microliters of urine diluted 1:20 with kit dilution solution containing d5-EtG and d5-EtS internal standards
• Direct injection of 15 μL into the UHPLC system

Liquid Chromatography

• Thermo Scientific Vanquish Duo UHPLC (direct-injection channel)
• Analytical column and mobile phases supplied by RECIPE
• Gradient runtime: 6.0 minutes; column temperature: 40 °C

Mass Spectrometry

• Thermo Scientific Q Exactive Plus hybrid quadrupole-Orbitrap MS
• Heated electrospray ionization in negative mode (HESI‐II source)
• Data acquisition: FullMS at 70,000 resolving power and PRM at 17,500 resolving power
• Processing with Thermo Scientific TraceFinder 5.1

Key Results and Discussion

The method demonstrated a linear response (1/x weighting) across calibration ranges of 81.7–9,368 ng/mL for EtG and 29.1–4,953 ng/mL for EtS. Lower limits of quantification (LLOQs) were 4.08 ng/mL for EtG and 14.6 ng/mL for EtS in both FullMS and PRM modes. Accuracy (bias within ±15%, ±20% at LLOQ) and trueness (bias within ±11%) were confirmed using internal quality controls and certified external controls. No carryover was observed. Intra-assay precision (%CV) remained below 6.3% (FullMS) and 3.3% (PRM), while inter-assay precision stayed under 10.0% (FullMS) and 5.1% (PRM), indicating excellent repeatability and reproducibility.

Benefits and Practical Applications

• High selectivity and sensitivity through Orbitrap HRAM technology
• Simple dilute-and-shoot protocol reduces preparation time and potential errors
• Simultaneous FullMS and PRM acquisition enhances confidence in qualitative and quantitative results
• Fast 6-minute chromatographic run supports high sample throughput

Future Trends and Potential Applications

Advances may include integration of online sample cleanup to further automate processing, expansion to multi-analyte panels for broader toxicology screening, and miniaturization of high-resolution MS for point-of-care testing. Machine learning–driven data analysis could enhance detection of low-level biomarkers and streamline method development.

Conclusion

The described LC-HRAM MS workflow provides a rapid, accurate, and precise solution for EtG and EtS quantification in urine. Its simple sample preparation, robust instrumentation, and dual acquisition strategy meet the requirements of clinical research laboratories for reliable alcohol biomarker analysis.