Synthesis, formation, and occurrence of contaminants in biotechnologically manufactured L-tryptophan.

The pattern of contaminants in pharmaceutical and feed grade L-tryptophan (Trp) was investigated in a market survey of 22 lots of 6 different manufacturers. To date, 5 case associated contaminants in Showa Denko tryptophan (SD-Trp) known to cause the autoimmune disease eosinophilia-myalgia syndrome (EMS) have been structurally elucidated: 3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrroloindole-2-carboxylic acid (PIC), an indoline compound, is one of the most abundant degradation compounds of unbound Trp during oxidative treatment. 2-(3-indolylmethyl)-L-tryptophan (IMT) and 2-(2-hydroxyindoline) -tryptophan (HIT) are both 2-substituted Trp-derivatives. IMT was synthesized by the reaction of Trp and indole-3-methanol or indole-3-acetaldehyde, respectively. From this finding it is proposed that Trp-metabolites can decompose under formation of transitional, mesomerism-stabilized cations that react with excess Trp to yield 2-substituted Trp derivatives. The decomposition of Trp-metabolites could be induced by elevated or low pH-values that occur during the downstream processing of the Trp fermentation broth. IMT was detected in pharmaceutical-grade and feed-grade Trp in amounts of <20-1,400mg/kg. 1,1′-Ethylidenebis-(L-tryptophan) (EBT) is formed from acetaldehyde and Trp under acidic conditions and serves as a marker for EMS-suspicious Trp. 3-(Phenylamino)alanine (PAA) is the only not Trp derived case associated contaminant. Low amounts of PAA (20mg/kg) could be detected in feed-grade Trp of one manufacturer. Non-EMS correlated 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acids of Trp and formaldehyde, acetaldehyde and indole-3-acetaldehyde could be detected in the examined Trp raw materials (<10-13,500 mg/kg).