Abstract: Ethyl Glucuronide (EtG) and Ethyl Sulfate (EtS) are established biomarkers for alcohol consumption. The analysis of these compounds poses challenges due to their polar nature, making them challenging to retain by reversed phase chromatography. Additionally, isobaric matrix interferences in urine must be fully resolved from the analytes to obtain accurate data.
In this work, a rapid LC-MS/MS method was developed that provides excellent resolution between the matrix interferences and the analytes of interest. The goal of this study was to establish a robust method capable of retaining both EtG and EtS while resolving urinary matrix interferences without the need for time consuming column pre-conditioning.
Synthetic urine was fortified with EtG and EtS from 50-1,000 ng/mL. Quality control (QC) samples were prepared using six lots of human urine, including samples from kidney disease and liver disease patients. Human urine was diluted 20-fold in water fortified with 0.1% formic acid. The samples were vortexed and centrifuged prior to injection on a Force Biphenyl column (100 x 3 mm, 3 μm). The mobile phases used were 0.1% formic acid in both water and methanol and the chromatographic separation was achieved under gradient conditions.
The developed method successfully resolved matrix interferences from the analytes of interest and no significant matrix interferences were observed across all QC urine lots. Linearity was demonstrated for both analytes with r² ≥ 0.99, and the method exhibited acceptable intra- and inter-day precision and accuracy.
The Force Biphenyl column offered improved robustness over multi-mode columns, which often require extensive pre-conditioning. In contrast, the Force Biphenyl allowed immediate use without compromising performance. It also provides flexibility for analyzing a broad range of compounds, including drugs of abuse and novel psychoactive substances, reducing the need for column changes.
