Contamination of commercially available L-tryptophan by related substances

In 1989 a new autoimmune disease, the eosinophilia myalgia syndrome (EMS), was traced back to the intake of L-tryptophan (Trp) of a single manufacturer, Showa Denko (SD). In an epidemiological study, six minor contaminants (10–1,000 mg/kg Trp) were related to the occurrence of EMS. In this study the pattern of contaminants in pharmaceutical- and feed-grade Trp was investigated in a market survey of 21 lots of Trp raw materials of six different manufacturers, as well as one bioconversion broth, and four tablets. For total Trp-related impurities all UV220 nm detectable substance peaks separated from Trp by RP-HPLC were calculated as N -acetyl-tryptophan. In all samples 4-,5-, 6-, and 7-OH-Trp, 1,2,3,4-tetrahydro-s-carboline-3-carboxylic acid (THCC), 1-methyl-1,2,3,4-tetrahydro-s-carboline-3-carboxylic acid (MTHCC) and 1-(3-indolylmethyl)-1,2,3,4-tetrahydro-s-carboline-3-carboxylic acid (IMTHCC) as well as 2-[2,3-dihydroxy-1-(3-indolyl)-propyl]- L- tryptophan (dhPIT) were determined, since the last four are known to be major contaminants in biotechnologically manufactured Trp. Additionally, four EMS-related substances were been analysed: 3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrroloindole-2-carboxylic acid (PIC), 2-(3-indolylmethyl)- L- tryptophan (IMT), 1,1'-ethylidenebis-( L- tryptophan) (EBT) and 3-(phenylamino)alanine (PAA). EBT was only detectable in a single SD-Trp sample known to be EMS associated. Low amounts of PAA (20 mg/kg) were detected in feed-grade Trp of one manufacturer as well as in SD-Trp. IMT was found in small amounts in recent pharmaceutical-grade Trp (10–25 mg/kg Trp), but in higher amounts in feed-grade Trp samples (120–1,400 mg/kg) as well as in pharmaceutical-grade Trp and Trp tablets manufactured prior to 1989 (70–660 mg/kg). PIC is a primary oxidation product of Trp; thus we detected 4–50 mg/kg of the earlier-eluting PIC diastereomer in recent pharmaceutical-grade Trp and up to 2,500 mg/kg in feed-grade Trp. Non-EMS correlated THCC, MTHCC, IMTHCC, and dhPIT proved to be the main contaminants in amounts of <10–13,500 mg/kg.