Ethanol Metabolites by Paper Spray Ionization: Method Development in Negative Ion Mode

Importance of the Topic

Paper spray ionization is a rapid, direct mass spectrometry technique that eliminates complex sample preparation and chromatography. Detecting ethanol metabolites ethyl glucuronide (EtG) and ethyl sulfate (EtS) in urine extends the diagnostic window for alcohol consumption beyond the short detection time of ethanol itself.

Study Objectives and Overview

This work aimed to develop and validate a negative-ion mode paper spray MS method for screening EtG and EtS in urine. The goal was to enable fast qualitative and quantitative analysis using Orbitrap mass spectrometry without liquid chromatography.

Methodology

Urine samples were spiked with EtG and EtS and corresponding deuterated internal standards. After dilution with methanol, centrifugation, and calibration curve preparation (5–1000 ng/mL), 8 µL aliquots were applied to paper cartridges. Samples dried for 10 min and were extracted with 100% methanol containing 100 ppm acetic acid. Analysis used full-scan MS at 70 000 resolving power and data-dependent MS/MS at 35 000 on Q Exactive Focus or Plus instruments.

Used Instrumentation

• Velox 360 paper spray system (Prosolia)
• Thermo Scientific Q Exactive Focus MS and Q Exactive Plus MS
• Thermo Scientific TraceFinder and Xcalibur software

Main Results and Discussion

• EtS anion ionized efficiently under negative mode with excellent linearity (R² ≈0.97) and reproducibility across 5–1000 ng/mL.
• EtG showed poorer reproducibility (R² ≈0.91) and lower signal stability, indicating potential ionization inefficiencies or interferences.
• Analysis of positive donor urine confirmed EtS detection; EtG signals varied widely, suggesting method refinement is needed.
• A single 40 s electrospray period supported both high-resolution full scans and multiple MS/MS events from one paper spot.

Benefits and Practical Applications

• Removes need for sample cleanup and chromatography, reducing analysis time to seconds per spot.
• Enables high-throughput forensic screening of urine for alcohol consumption markers.
• Internal standards and cellulose substrate support quantitative EtS measurement.

Future Trends and Applications

• Investigate alternative paper substrates or surface treatments to improve EtG ionization efficiency.
• Acquire MS/MS data at higher resolving power (70 000 FWHM) to minimize interferences.
• Expand negative-mode paper spray approaches to other conjugated metabolites in clinical and forensic toxicology.

Conclusion

A negative-ion mode paper spray MS method was successfully developed for rapid screening of ethyl sulfate in urine using Q Exactive mass spectrometers. Further optimization is required for reliable quantitation of ethyl glucuronide. This technique offers a fast, high-resolution alternative to LC-MS for forensic alcohol testing.

References

• Jalbert et al. Journal of Chromatography A 1228 (2012) 51–56
• Manicke et al. International Journal of Mass Spectrometry 300 (2011) 123–129