Extraction of Ethyl Glucuronide (EtG) from Urine Using EVOLUTE AX for LC–MS-MS Analysis - - Chromatography Online
Extraction of Ethyl Glucuronide (EtG) from Urine Using EVOLUTE AX for LC–MS-MS Analysis

Lee Williams, Rhys Jones, Helen Lodder, Steve Jordan, Claire Desbrow, and Gary Dowthwaite, Biotage GB Limited

Ethyl glucuronide (EtG) is an ethanol metabolite formed by the glucuronidation of the parent molecule. The presence of EtG in urine is a useful and sensitive bio-marker of alcohol intake in forensic toxicology. EtG can be detected at very low levels and being a metabolite it is useful in distinguishing between ingested ethanol and that which is adsorbed through the skin from ethanol containing products such as alcohol based hand washes. Rapid and reliable methods with robust extraction protocols are essential for analysis and quantitation of EtG from various matrices.

Extraction Conditions

This application note outlines the procedure using the 25 mg 96-well plate format (part #603-0025-P01) but other formats are available, method parameters and dilution factors have been optimized to maximize recoveries and minimize ion suppression.

Sample: urine (50 μL) spiked at 500 ng/mL with EtG.
Sample pre-treatment: urine (50 μL) in acetonitrile (950 μL).
Plate conditioning: methanol (1 mL)
Plate equilibration 1: water (1 mL)
Column equilibration 2: acetonitrile (1 mL)
Sample loading: pre-treated sample (1 mL)
Interference elution 1: water (1 mL)
Interference elution 2: acetonitrile (1 mL)
Analyte elution: acetonitrile:water:formic acid, (1 mL, 95:4:1, v/v)
Post extraction: evaporate to dryness and reconstitute with water/methanol (200 μL, 80:20, v/v) for LC–MS-MS analysis.

The extracted samples were then run on a Waters Acquity UPLC (Waters Assoc., Milford, Massachusetts) with an Acquity UPLC BEH C18 column (1.7 μm, 50 2.1 mm id) (Waters Assoc.) at 40 C. Mobile phase was isocratic, 0.1% (v/v) formic acid aq/methanol (80:20, v/v) at a flow rate of 0.5 mL/min. The detector was a Premier XE triple quadrupole mass spectrometer (Waters Assoc., Manchester, UK) equipped with an electrospray interface for mass analysis. Negative ions were acquired in the multiple reaction monitoring mode (MRM); 21 > 84.9 (quantifier), 221.0 > 74.9 (qualifier).


Figure 1: EtG extraction recoveries from urine using EVOLUTE AX.
As demonstrated in Figure 1 recoveries over 80% with RSDs below 10% were achieved across the range of EVOLUTE AX formats and sample volumes using this application.


The method demonstrates robust and effective extraction of EtG not only using a 25 mg 96 well-plate but also the optimization is scalable for sample volumes ranging from 50 μL up to 1 mL across format changes from plate to column. As the sample volume increases it is in fact easier to obtain higher recovery rates of EtG, possibly because of the lack of a concentration step.


(1) L. Williams, et. al. Extraction of Ethyl glucuronide (EtG) from urine using EVOLUTE AX. Application Note AN 705. (available from http://www.biotage.com/).

Biotage, LLC
10430 Harris Oarks Blvd., Suite C., Charlotte, NC 28269
Website: http://www.biotage.com/


blog comments powered by Disqus
LCGC E-mail Newsletters
Global E-newsletters subscribe here:



Column Watch: Ron Majors, established authority on new column technologies, keeps readers up-to-date with new sample preparation trends in all branches of chromatography and reviews developments. LATEST: When Bad Things Happen to Good Food: Applications of HPLC to Detect Food Adulteration

Perspectives in Modern HPLC: Michael W. Dong is a senior scientist in Small Molecule Drug Discovery at Genentech in South San Francisco, California. He is responsible for new technologies, automation, and supporting late-stage research projects in small molecule analytical chemistry and QC of small molecule pharmaceutical sciences. LATEST: HPLC for Characterization and Quality Control of Therapeutic Monoclonal Antibodies

MS — The Practical Art: Kate Yu brings her expertise in the field of mass spectrometry and hyphenated techniques to the pages of LCGC. In this column she examines the mass spectrometric side of coupled liquid and gas-phase systems. Troubleshooting-style articles provide readers with invaluable advice for getting the most from their mass spectrometers. LATEST: Radical Mass Spectrometry as a New Frontier for Bioanalysis

LC Troubleshooting: LC Troubleshooting sets about making HPLC methods easier to master. By covering the basics of liquid chromatography separations and instrumentation, John Dolan is able to highlight common problems and provide remedies for them. LATEST: How Much Can I Inject? Part I: Injecting in Mobile Phase

More LCGC Columnists>>

LCGC North America Editorial Advisory Board>>

LCGC Europe Editorial Advisory Board>>

LCGC Editorial Team Contacts>>

Source: Special Issues,
Click here