Amphi-enterobactin is an amphiphilic siderophore isolated from a variety of microbial Vibrio species. Like enterobactin, amphi-enterobactin is a triscatecholate siderophore; however, it is framed on an expanded tetralactone core comprised of four L-Ser residues, of which one L-Ser is appended by a fatty acid and the remaining L-Ser residues are appended by 2,3-dihydroxybenzoate (DHB). Fragments of amphi-enterobactin composed of 2-Ser-1-DHB-FA and 3-Ser-2-DHB-FA have been identified in the supernatant of Vibrio campbellii species. The origin of these fragments has not been determined, although two distinct isomers could exist for 2-Ser-1-DHB-FA and three distinct isomers could exist for 3-Ser-2-DHB-FA. The fragments of amphi-enterobactin could originate from hydrolysis of the amphi-enterobactin macrolactone, or from premature release due to an inefficient biosynthetic pathway. Unique masses in the tandem MS analysis establish that certain fragments isolated from the culture supernatant must originate from hydrolysis of the amphi-enterobactin macrolactone, while others cannot be distinguished from premature release during biosynthesis or hydrolysis of amphi-enterobactin.