Structure determination of disease associated peak AAA from L-Tryptophan implicated in the eosinophilia-myalgia syndrome

The eosinophilia-myalgia syndrome (EMS) outbreak of 1989 that occurred in the USA and elsewhere was caused by the ingestion of L-Tryptophan (L-Trp) solely manufactured by the Japanese company Showa Denko K.K. (SD). Six compounds present in the SD L-Trp were reported to be case-associated contaminants. However, one of these compounds, Peak AAA has remained structurally uncharacterized, despite the fact that it was described as the only statistically significant (p = 0.0014) contaminant. Here, we employ on-line microcapillary-high performance liquid chromatography-electrospray ionization mass spectrometry (LC-MS), and tandem mass spectrometry (MS/MS) to determine that Peak AAA is in fact two structurally related isomers. Peak AAA(1) and Peak AAA(2) differed in LC retention times, and were determined by accurate mass-LC-MS to both have a protonated molecular ion (MH+) of mass 343.239 Da (Da), corresponding to a molecular formula of C21H30N2O2, and possessing eight degrees of unsaturation (DoU) for the non-protonated molecule. By comparing the LC-MS and LC-MS-MS retention times and spectra with authentic synthetic standards, Peak AAA(1) was identified as the intermolecular condensation product of L-Trp with anteiso 7-methylnonanoic acid, to afford (S)-2-amino-3-(2((S,E)-7-methylnon-1-en-1-yl)-1H-indol-3-yl) propanoic acid. Peak AAA(2) was determined to be a condensation product of L-Trp with decanoic acid, which produced (S)-2-amino-3-(2-((E)-dec-1-en-1-yl)-1H-indol-3-yl) propanoic acid.