Separation and isolation of trace impurities in L-tryptophan by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) profiling method was developed to separate trace impurities in L-tryptophan products associated with the eosinophilia-myalgia syndrome (EMS) epidemic. The test portion was dissolved in water, and the solution was filtered and chromatographed on a silica-based C18 reversed-phase HPLC column by using linear gradient elution with water and acetonitrile-water (80:20); both solvents contained 0.1% trifluoroacetic acid for ion-pairing. The method was used to profile 200 test samples from six manufacturers of L-tryptophan. The method was modified to include the use of a C18 disposable cartridge to retain the 1,1'-ethylidene-bis(L-tryptophan) (peak E, peak 97 or EBT), the impurity most strongly associated with EMS, and to remove the L-tryptophan before HPLC separation and quantitation. Recoveries of EBT added to test portions (2 micrograms/g and above) averaged 80%.