High-performance liquid chromatographic determination of 1,1'-ethylidenebis(L-tryptophan) in L-tryptophan...

In studies to determine the cause or causes of the eosinophilia myalgic syndrome (EMS) and to monitor the purity of L-tryptophan preparations, an HPLC method has been developed for determining 1,1'-ethylidenebis(L-tryptophan) (EBT) in L-tryptophan (W) preparations. The W preparations are extracted with 0.1% trifluoroacetic acid (TFA) and filtered, and the EBT is purified by passage through a Sep-Pak C18 cartridge. The cartridge is washed with water and 6% acetonitrile in water, and EBT is eluted with methanol. The water-diluted eluate is then chromatographed on a silica-based, reversed-phase HPLC column with a gradient of water and 80% acetonitrile, both solvents containing 0.1% TFA. EBT absorbance is measured at 280 nm. The average recovery of EBT from L-tryptophan powder, spiked over the range 1.2-4.8 micrograms/g, was 91%. The limit of determination was approximately 0.6 micrograms/g. Sixteen test samples of W products manufactured by the company to which most of the cases of EMS have been traced contained > 70 micrograms of EBT/g. Three nonpatient-related test samples either did not contain EBT or contained < 2 micrograms of EBT/g.