Ethyl sulfate (EtS)-a new direct marker for ethanol intake besides ethyl glucuronide (EtG) and others-was detected in urine samples by electrospray ionization tandem mass-spectrometry (LC-ESI-MS/MS). Ethyl sulfate sodium salt was used for method development, yielding a precursor [ After addition of D 5 -EtS and D 5 -EtG, urine samples were analyzed by direct injection into the gradient LC-MS/MS system. Analysis was performed in accordance with forensic guidelines for confirmatory analysis using one precursor and two product ions. EtS has been detected (in addition to EtG) in the urine samples of nine volunteers after drinking sparkling wine containing between 9 and 49 g of ethanol. Both EtS and EtG could be detected up to 36 h after consumption of alcohol. The excretion profile was found to be similar to that of EtG. No EtS was found in teetotalers' urine samples. Method validation parameters are presented. EtS was stable in urine upon storage up to twenty days at room temperature. In addition to EtG, EtS can be used to detect recent alcohol consumption, thus providing a second marker for the time range of up to approximately one day after elimination of ethanol from urine samples , and its detection in rat urine after dosing with 35 S-sulfate and ethanol, was performed by thin-layer chromatography and by autoradiographic detection on X-ray films [2]. Lung tissue was found to have the ability to metabolize ethanol via glucuronidation [3] and by sulfation [4,5]. Later, Manautou and Carlson compared the hepatic and pulmonary metabolism via glucuronidation and sulfation in rats and rabbits [6]. Meanwhile, for sulfate conjugation a superfamily of cytosolic sulfotransferases has been described that shows a genetic polymorphism [7]. The conjugation of aliphatic alcohols in humans has been mentioned by Bonte et al. when investigating metabolites of higher aliphatic alcohols [8]; detection was performed indirectly via cleavage by sulfatase followed by analysis of the alcohols. However, no direct analytical method to detect and quantify EtS as a marker for recent ethanol consumption by humans has been available until very recently. Parallel to our work, Helander and Beck have developed an LC-ESI-MS assay using single-quadrupole mode and D 5 -ethyl glucuronide as internal standard for quantitation of EtS in urine samples [9]. However, selected ion monitoring of the deprotonated molecule with a single-stage quadrupole MS cannot be applied to forensic samples as the only method for compound detection, since it does not fulfill forensic criteria to prove compound identity [10]. When the results of urine drug testing can affect an individu-