Sensitive Quantification of the Ethylene Oxide Hemoglobin Adduct <i>N</i> -(2-Hydroxyethyl)- <scp>l</scp> -valine from Minimal Blood Volumes

-(2-hydroxyethyl)-l-valine (HE-V). However, current methods require large blood volumes, limiting small-animal exposure studies and reliable detection of endogenous background levels of HE-V. We optimized a highly sensitive analytical workflow including hemoglobin extraction from microliter-scale blood samples, HE-V purification, and targeted LC-MS/MS quantitative analysis. This workflow reliably quantifies endogenous HE-V levels of unexposed mice from only 50 μg of purified hemoglobin (10 μL of blood) and is demonstrated to detect significant HE-V increases at EtO exposures as low as 0.05 ppm. Our platform enables practical EtO biomonitoring in volume-constrained experimental and environmental settings.