BackgroundThere is controversy regarding the active agent responsible for alcohol addiction. The theory that ethanol (EtOH) itself was the agent in alcohol drinking behavior was widely accepted until acetaldehyde (AcH) was found in the brain. The importance of AcH formation in the brain is still subject to speculation due to the lack of a method to accurately assay the AcH levels directly. A highly sensitive gas chromatography mass spectrometry (GC‐MS) method to reliably determine AcH concentration with certainty is needed to address whether neural AcH is indeed responsible for increased alcohol consumption.MethodsA headspace gas chromatograph coupled to selected‐ion monitoring MS was utilized to develop a quantitative assay for AcH and EtOH. Our GC‐MS approach was carried out using a Bruker Scion 436‐GC SQ MS.ResultsOur approach yields limits of detection of AcH in the nanomolar range and limits of quantification in the low micromolar range. Our linear calibration includes 5 concentrations with a least‐square regression greater than 0.99 for both AcH and EtOH. Tissue analyses using this method revealed the capacity to quantify EtOH and AcH in blood, brain, and liver tissue from mice.ConclusionsBy allowing quantification of very low concentrations, this method may be used to examine the formation of EtOH metabolites, specifically AcH, in murine brain tissue in alcohol research.