LC-MS/MS Analysis of EtG and EtS in Dilute Urine on the TSQ Endura Triple Quadrupole MS

Significance of the topic

Alcohol biomarkers like ethyl glucuronide (EtG) and ethyl sulfate (EtS) provide extended detection windows for alcohol consumption due to their longer biological half-lives. However, their high polarity challenges reversed-phase LC, causing poor retention and co-elution near the solvent front. Developing a reliable LC-MS/MS assay for EtG and EtS in urine is critical for accurate forensic and clinical toxicology analyses.

Aims and study overview

The primary objective was to establish an HPLC-MS/MS method capable of quantifying EtG with a limit of quantitation (LOQ) of 100 ng/mL and EtS with an LOQ of 50 ng/mL using only simple urine dilution. The method targets high sensitivity, broad dynamic range, and streamlined sample preparation for forensic applications.

Methodology

• Sample preparation: 25 µL urine mixed with deuterated internal standards (EtG-d5, EtS-d5), diluted to 500 µL with water; 30 µL injected.
• Chromatography: Dionex UltiMate 3000 RSLC, Hypersil GOLD column (50×3 mm, 5 µm), gradient elution with 5 mM dihexylammonium acetate in water/acetonitrile, flow rate 1 mL/min, 5 min total run.
• Data processing: Acquisition and quantification using Thermo TraceFinder software.

Instrumentation

• LC system: Thermo Scientific Dionex UltiMate 3000 RSLC.
• Mass spectrometer: Thermo Scientific TSQ Endura triple quadrupole with Ion Max NG source and HESI-III probe.
• Column: Thermo Scientific Hypersil GOLD (50×3 mm, 5 µm).

Main results and discussion

• Linearity: EtG 50–50,000 ng/mL; EtS 25–50,000 ng/mL with calibration bias within ±15%.
• Retention and peak shape improved by ion-pairing reagent, reducing matrix effects.
• Precision and accuracy: Inter-assay bias ≤3.3% and RSD ≤12% at low QC levels; consistent intra-assay performance.
• Detection limits: 50 ng/mL for EtG; 25 ng/mL for EtS.
• Matrix effects: Stable internal standard recovery across 23 urine lots demonstrated method robustness.

Benefits and practical application

• Minimal sample cleanup accelerates workflow and reduces sample loss.
• High sensitivity and wide dynamic range accommodate both low-level and high-level analyte quantification.
• Simplicity of dilution-based preparation supports high-throughput forensic and clinical laboratories.

Future trends and potential applications

• Automation: Integration with automated dilution systems to further enhance throughput.
• Matrix expansion: Adaptation for other biological specimens such as blood or hair for comprehensive alcohol exposure monitoring.
• Panel development: Combined analysis of multiple alcohol and drug biomarkers for detailed consumption profiling.

Conclusion

The described LC-MS/MS method delivers sensitive, accurate, and reproducible quantification of EtG and EtS in diluted urine. Using ion-pairing chromatography and minimal sample preparation, it meets forensic requirements for detection limits, precision, and dynamic range while simplifying laboratory workflows.

References

• Van Natta K, Kozak M. LC-MS/MS Analysis of EtG and EtS in Dilute Urine on the TSQ Endura Triple Quadrupole MS. Thermo Fisher Scientific; 2016.
• TCI America. Dihexylammonium Acetate Product Information.
• Fisher Chemical. Reagent Grades for Water and Acetonitrile; Safety Data.