Introduction: 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, there are isobaric matrix interferences in urine that require full chromatographic resolution from the analytes to obtain accurate data. Disease state samples provide an additional challenge due to the potential of unique interferences. In this work, a rapid LC method was developed that demonstrated excellent resolution between the matrix interferences and the analytes of interest.
Objectives: This study aimed to develop a robust LC-MS/MS method capable of retaining both EtG and EtS while resolving urinary matrix interferences in both normal and disease state patient samples.
Methods: Calibrators were prepared in synthetic urine fortified with EtG and EtS from 50-1000 ng/mL. Quality control (QC) samples were prepared using six lots of human urine, including three from single-donor disease state patients (kidney disease, liver disease, diabetes). Samples were diluted 20-fold in water fortified with 0.1% formic acid. The samples were then vortexed and centrifuged prior to a 10 μL injection on a Force Biphenyl column (100 x 3 mm, 3 μm) at 30 °C. The mobile phases used were water and methanol, both modified with 0.1% formic acid (v/v), and the chromatographic separation was achieved under gradient conditions starting at 100% aqueous. Precision and accuracy experiments were performed on a Shimadzu LCMS-8060 triple quadrupole using electrospray ionization in negative ion mode.
Results: The developed method successfully resolved matrix interferences from the analytes of interest. No significant matrix interferences were observed across the QC urine lots, including those in disease state samples. Linearity was demonstrated for both analytes with r² ≥ 0.99, and the method exhibited acceptable (+/-10%) intra- and interday precision and accuracy. The use of a fully porous Biphenyl column enabled adequate retention for these polar analytes to separate matrix interferences.
Discussion: A rapid and reliable LC-MS/MS method was developed for the quantitation of EtG and EtS in urine using a fully porous Biphenyl column. The method was verified across multiple lots of human urine and disease state samples, offering a robust and versatile solution for clinical and forensic applications. 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.
