ONLINE HPLC TANDEM MASS-SPECTROMETRY STRUCTURAL CHARACTERIZATION OF CASE-ASSOCIATED CONTAMINANTS OF L-TRYPTOPHAN IMPLICATED WITH THE ONSET OF EOSINOPHILIA-MYALGIA-SYNDROME

The eosinophilia-myalgia syndrome (EMS) outbreak that occurred in the USA in 1989 was caused by the intake of L-tryptophan (Trp) produced fr om one manufacturer, Showa Denko K.K. of Japan. Six compounds present in the Trp were reported to be case-associated contaminants. However, three of these compounds, Peaks C, FF and AAA have remained unidentifi ed. Here, we successfully employ on-line HPLC-electrospray ionization multistage mass spectrometry to structurally characterize Peak C and P eak FF. Peak C was determined by accurate mass-LC-MS to have a protona ted molecular ion MH+ = 221.0919 with an empirical formula of C11H13N2 O3. By comparing the LC-MS-MS spectra with authentic 5-OHTrp and other structurally similar compounds, as well as considering the chemical r eactivity of the indole ring, the structure of Peak C was consistent w ith ,8,8a-hexahydropyrrolo-[2,3-b]-indole-2-carboxylic acid. Peak FF w as also subjected to accurate mass-LC-MS and shown to have MH+ = 338.1 524, corresponding to an empirical formula of C19H20N3O3. Comparison o f the LC-MS-MS and LC-sCID-MS-MS of spectra derived from Peak FF with a previously characterized contaminant of Trp, namely P31, was consist ent with Peak FF being 2-(2-hydroxy indoline)-Trp. Unlike the majority of the contaminants identified in EMS implicated tryptophan, both Pea ks C and FF possess an indoline ring. This is significant since a case -associated contaminant found in 5-hydroxy-Trp also contains an indoli ne ring, and the chemical reactivity of this ring system may possibly play a role in the etiology of EMS. (C) 1998 Published by Elsevier Sci ence Ireland Ltd. All rights reserved.