MSACL 2022 Abstract

Quantitative LC-MS/MS Analysis of Ethyl Glucuronide and Ethyl Sulfate in Urine Using Poroshell Charged Surface C18 Column

Natalie Rasmussen, Andre Szczesniewski
Agilent Technologies, Santa Clara, CA

Natalie Rasmussen, BS Biology (Presenter) Agilent Technologies

Presenter Bio: Natalie graduated from the University of Utah with a BS in biology in 2004.

She began work at ARUP Laboratories in Salt Lake City in 2005. She worked in a clinical toxicology lab, preparing and analyzing meconium, urine, and serum samples by enzyme immunoassay and mass spectrometry (LC-MS/MS and LC-(Q)TOF-MS).

After 8 years of routine bench work, she moved into the research and development area. There she coordinated a head injury study with the University of Utah football team, developed and improved mass spec-based diagnostic tests, collaborated in research studies, presented at national conferences, and contributed to peer-reviewed publications and application notes.

Natalie joined Agilent as a LC Applications Scientist in August 2017, where she primarily focuses on small molecule LC columns.

Relevant Financial Disclosures (within past 24 months, reported on Jul 17, 2025) Stock/Bonds Agilent Technologies Salary Agilent Technologies Conflict mitigation NOT REQUIRED.

Introduction:
Ethyl glucuronide (EtG) and ethyl sulfate (EtS) biomarkers are formed by the liver after ethanol ingestion. Eliminated by the kidneys, they can be detected in urine for approximately 2-80 hours post-imbibing. Due to the polar nature of these metabolites, they are difficult to retain on a reversed-phase column. This study tested if a hybrid end-capped charged surface C18 column would retain them well enough for detection and quantification.

Methods:
Urine was spiked with standards made from a working stock solution. Calibrators, controls, and samples were spiked with internal standard, diluted 1:50 in mobile phase A, and injected on an Agilent InfinityLab Poroshell 120 CS-C18 column and analyzed in negative ion mode by LC/MS/MS. Total run time was approximately 4.5 minutes.

Data:
The two compounds were resolved from each other and well-retained with k’ of 2.2 for EtG and 3.5 for EtS using a 3-minute gradient. Through a series of experiments with different dilutions, it was determined that a high dilution (1:50) and low injection volume (2µL) produced the best peak shape and signal-to-noise ratio, while eliminating a peak that interfered with EtG. Over 800 injections, retention times and peak areas were extremely stable. The calibration curves injected in triplicate were linear from a range of 100-10,000 ng/mL with an R2= >0.998 with RSDs less than 2%.

Conclusion:
While further experiments are needed to determine column lifetime and method robustness, preliminary data indicate this method would work well in a high-throughput environment due to its short run time and simple sample preparation.

For Research Use Only. Not for use in diagnostic procedures.