HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC DETERMINATION OF 1,1'-ETHYLIDENEBIS(L-TRYPTOPHAN) IN L-TRYPTOPHAN PREPARATIONS

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 C-18 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 mu g/g, was 91%. The limit of determination was similar to 0.6 mu g/g. Sixteen test samples of W products manufactured by the company to which most of the cases of EMS have been traced contained >70 mu g of EBT/g. Three nonpatient-related test samples either did not contain EBT or contained <2 mu g of EBT/g.