A Rapid Dilute and Shoot - Flow Injection MRM Method for the Quantification of EtG in Urine

Quantification of ethyl glucuronide (EtG, a metabolite and biomarker of ethanol) in urine is of great interest in both forensic and clinical applications. Currently, a single run of LC-MS/MS can quantify the vast majority of drugs and abused substances included in the comprehensive urine drug testing (UDT) panel, but EtG must be quantified in a separate run because EtG is polar, limiting the throughput of current MS-based methods. In this study, we developed a simple and fast MRM method to quantify EtG in urine without LC separation. Briefly, a urine sample containing EtG is first diluted 20 times, followed by direct flow-injection of the diluted sample to the ESI source. Quantification of EtG was achieved by multiple reaction monitoring (MRM) operated in the negative mode with stable isotope labeled EtG as internal standard. The method was fully validated and has a dynamic range of 50-2000ng/mL (R2>0.998). The coefficients of variation and relative errors for interand intra-assay studies were in the range of 2.4-4.2%, respectively.In addition, a robustness test was performed with >1200 injections of urine samples and no significant deteriorations in sensitivity and specificity were observed. This new method has a total run time of only 2 min per sample, enabling analysis of >700 samples per day by one single mass spectrometer.