Ultrafast confirmation and quantitation of alcohol metabolite markers using the EVOQ Elite TQ coupled to the Elute UHPLC

Significance of the Topic

Ethyl glucuronide (EtG) and ethyl sulfate (EtS) serve as sensitive biomarkers for recent alcohol exposure, extending detection beyond the short-lived presence of ethanol itself. Their reliable identification in urine is crucial across forensic, clinical and workplace testing scenarios, where assessing abstinence or alcohol consumption history influences legal decisions, fitness-to-drive evaluations and compliance monitoring.

Objectives and Study Overview

This work aimed to establish an ultrafast, precise and robust assay for simultaneous quantitation of EtG and EtS in human urine. Utilizing a novel ultra-high-performance liquid chromatography (UHPLC) system coupled with a triple quadrupole mass spectrometer, the study validated linearity, accuracy and precision across clinical relevant ranges and applied the method to both proficiency testing materials and real donor samples.

Methodology and Instrumentation

Sample Preparation

• Urine calibrators (six levels) and quality control materials were obtained commercially.
• Clinical specimens included blinded proficiency samples and post-consumption urine from two volunteer subjects, sampled at 3 and 24 hours after ingesting defined alcohol quantities.
• Protein precipitation was performed by adding methanol and isotopically labeled internal standards to centrifuged urine, followed by supernatant evaporation and reconstitution in aqueous formic acid.

Used Instrumentation

• UHPLC: Bruker Elute system with a Restek Ultra Biphenyl column (2.1×100 mm, 3 μm) plus guard column.
• Mass Spectrometer: EVOQ Elite triple quadrupole in negative electrospray ionization (VIP H-ESI) mode.
• Mobile Phase: Water and methanol each with 2 mM ammonium formate and 0.1% formic acid under a fast gradient (2–80% organic in 1 minute).
• MRM Transitions: Monitored specific precursor/product ion pairs for EtG, EtS and their deuterated analogs.

Main Results and Discussion

The calibration curves for both EtG (80–9621 ng/mL) and EtS (27–4732 ng/mL) exhibited excellent linearity (r2 ≥ 0.999) and accuracy within ±8% bias. Quality control samples showed deviations below 10%. Precision assessments using proficiency testing materials delivered relative standard deviations (RSD) under 2.8% and results aligned within acceptance criteria. Real urine specimens collected 3 hours post-alcohol intake exceeded the upper calibration limits, confirming high sensitivity, while 24-hour samples still yielded quantifiable concentrations for both analytes. Chromatographic performance featured sharp peak shapes with full width at half maximum of 3–4 seconds, supporting rapid cycle times and high throughput.

Benefits and Practical Applications of the Method

This approach combines minimal sample preparation with an analytical cycle time of just three minutes, enabling laboratories to process large sample sets efficiently. The dual measurement of EtG and EtS mitigates false negatives due to microbial degradation and false positives from incidental ethanol exposure, providing greater diagnostic confidence for forensic and clinical settings.

Future Trends and Potential Applications

As UHPLC–MS/MS platforms continue to advance, further miniaturization of flow paths and integration of automated sample handling will likely reduce analysis time and solvent consumption. Expanding the assay to other biological matrices like hair or oral fluid can enhance monitoring windows. Moreover, multiplexed workflows incorporating additional phase II metabolites may offer comprehensive profiles of substance use and metabolism.

Conclusion

A rapid, reliable UHPLC–MS/MS method for quantifying EtG and EtS in urine was developed, demonstrating outstanding linearity, accuracy and precision. Its streamlined workflow and robust performance position it as a valuable tool for forensic toxicology and clinical alcohol monitoring.

Reference

No external literature citations were provided in the original document.