AbstractIn this paper we describe the application of electrospray time‐of‐flight mass spectrometry (ESI‐TOFMS) to structural elucidation of the fragment ions formed from a range of natural and synthetic allelochemical derivatives. The extensive mass spectrometric characterisation of ten non‐glucosylated benzoxazinone derivatives using this method is described here for the first time. The analytes include six naturally occurring 1,4‐benzoxazin‐3(4H)‐one derivatives, including the hydroxamic acids DIMBOA [2,4‐dihydroxy‐7‐methoxy‐2H‐1,4‐benzoxazin‐3(4H)‐one] and DIBOA [2,4‐dihydroxy‐2H‐1,4‐benzoxazin‐3(4H)‐one], lactams HBOA [2‐hydroxy‐2H‐1,4‐benzoxazin‐3(4H)‐one] and HMBOA [2‐hydroxy‐7‐methoxy‐2H‐1,4‐benzoxazin‐3(4H)‐one], benzoxazolinones BOA [benzoxazolin‐2(3H)‐one] and MBOA [6‐methoxy‐benzoxazolin‐2(3H)‐one] and four synthetic variations, 2′H‐DIBOA [4‐hydroxy‐2H‐1,4‐benzoxazin‐3(4H)‐one], 2′OMe‐DIBOA [2‐methoxy‐4‐hydroxy‐2H‐1,4‐benzoxazin‐3(4H)‐one], 2′H‐HBOA [2H‐1,4‐benzoxazin‐3(4H)‐one] and 2′OMe‐HBOA [2‐methoxy‐2H‐1,4‐benzoxazin‐3(4H)‐one]. Assignments of the mass spectral fragments were aided by elemental composition calculation results, comparison of structural analogues and background literature, and acquired knowledge regarding feasible structures for the compounds. The influence of substituents on the chemical reactivity of the compounds with respect to the observed MS behaviour over varying nozzle potentials is addressed and, through comparison of the structural analogues, generic fragmentation patterns have also been identified. Copyright © 2003 John Wiley & Sons, Ltd.