The presence of the cocaine pyrolysis product anhydroecgonine methyl ester (AEME, methylecgonidine) in plasma indicates the smoking of cocaine. The stability of this analyte in human plasma has not been studied. In the present investigation AEME and its hydrolysis product anhydroecgonine (AE, ecgonidine) were assayed in plasma and buffers using gas chromatography-mass spectrometry. It was found that 50% of AEME in human plasma was hydrolyzed to AE within 5 days at room temperature and within 13 days at 4 degrees C. Addition of esterase inhibitors such as sodium fluoride or echothiophate iodide reduced the hydrolysis significantly. Hydrolysis of AEME also occurred in buffers with pH values above 5, but the hydrolysis rate was significantly lower when compared with plasma and could be markedly increased by the addition of butyrylcholine esterase. The data suggest that AEME is degraded via two mechanisms: chemical hydrolysis at basic pH and enzymatic hydrolysis by butyrylcholine esterase. Therefore, proper storage conditions must be provided for the assay of AEME in plasma.