Alkyl glucamides (AGs) were analyzed by reversed‐phase liquid chromatography (LC) coupled to mass spectrometry with electrospray ionization (ESMS). Analytes were separated according to the chain length of two homologs, C12‐ and C14‐glucamide. Mass spectrometric detection in the positive ion mode exhibited higher overall sensitivity where, apart from fragments, different molecular and quasi‐molecular ions were obtained. However, application of the negative ion mode offered advantages in terms of reproducibility and extent of information when analyzing environmental samples. Therefore, a simple and sensitive analytical methodology was developed for the determination of AGs in municipal sewage treatment plant influent and effluent based on solid‐phase enrichment, LC separation and negative ion ESMS quantification. After preconcentration of 100 ml of aqueous sample, the recoveries using polymeric LiChrolut EN cartridges exceeded 89%. A quantification limit of 0.1 µg l−1 was achieved. Studies on the biodegradability and metabolic pathway of C10‐glucamide were carried out on a laboratory‐scale microbial test unit under aerobic conditions. A postulated metabolism including ω‐oxidation of the alkyl chain followed by subsequent β‐oxidations was checked by LC/ESMS. Identification and formation of ‘C4‐glucamide acid’ as a degradation intermediate was confirmed by mass spectrometric studies. Higher homolog acids such as C10‐, C8‐ and C6‐glucamide acids, which should be predicted precursors of C4‐glucamide acid, and any other metabolites, were not detectable, presumably owing to rapid breakdown. Copyright © 2000 John Wiley & Sons, Ltd.