Three different commercially available melatonin preparations were analyzed by on-line HPLC-electrospray ionization-tandem mass spectrometry. All three samples contained the same impurities at the approximately 0.1-0.5% level of parent melatonin. Based on accurate mass-HPLC-MS and tandem mass spectrometric analyses, two contaminants (both MH+ = 249) were identified as hydroxylation products of melatonin. One compound (MH+ = 265) was determined to be a C-2 oxidation product of hydroxymelatonin and a group of four regioisomers (MH+ = 477) were identified as melatonin-formaldehyde condensation products. These latter contaminants are structural analogues of the case-associated peak "E" found in L-tryptophan implicated in onset of eosinophilia-myalgia syndrome. The significance of these findings is discussed.